Modulation of Structure and Dynamics by Disulfide Bond Formation in

Supporting Information

for

Modulation of Structure and Dynamics by Disulfide Bond Formation in Unfolded States

Robert Silvers1, Friederike Sziegat1, Hideki Tachibana2, Shin-ichi Segawa3, Sara Whittaker4, Ulrich L. Günther4, Frank Gabel5, Jie-rong Huang5, Martin Blackledge5, Julia Wirmer-Bartoschek1, Harald Schwalbe1,* 1

Institute for Organic Chemistry and Chemical Biology, Center of Biomolecular Magnetic Resonance, Goethe University

Frankfurt, Max-von-Laue-Strasse 7, 60438 Frankfurt am Main, Germany 2

School of Biology-Oriented Science and Technology, Kinki University, Kinokawa, 649-6493, Japan

3

School of Science and Technology, Kwansei Gakuin University, Sanda, 669-1337, Japan

4

The Henry Wellcome Building for Biomolecular NMR Spectroscopy, School of Cancer Sciences, University of

Birmingham, Edgbaston, Birmingham B15 2TT, UK 5

Institut de Biologie Structurale Jean-Pierre Ebel, CEA-CNRS-UJF UMR 5075, 41 Rue Jules Horowitz, Grenoble 38027,

France

PAGE S1

EXPERIMENTAL SECTION

Constructs. Several lysozyme constructs were used. Native hen egg white lysozyme is synthesized in Gallus gallus (Chicken) as a precursor with an N-terminal signal peptide of 18 amino acids. The matured protein consists of 129 amino acids, where the N-terminal portion was cut. It has the following one-letter code: 10 20 30 40 50 60 KVFGRCELAA__AMKRHGLDNY__RGYSLGNWVC__AAKFESNFNT__QATNRNTDGS__TDYGILQINS 70 80 90 100 110 120 RWWCNDGRTP__GSRNLCNIPC__SALLSSDITA__SVNCAKKIVS__DGNGMNAWVA__WRNRCKGTDV 129 QAWIRGCRL

We used a construct with an additional N-terminal methionine for recombinant expression in E. coli, named 4SS-HEWL. Constructs and their mutations in respect to 4SS-HEWL are as follows: 0SS-HEWL 1SS64-80-HEWL 1SS76-94-HEWL 1SS6-127-HEWL 1SS30-115-HEWL 1SS30-115W62G-HEWL

C6A, C30A, C64A, C76A, C80A, C94A, C115A, C127A C6A, C30A, C76A, C94A, C115A, C127A C6A, C30A, C64A, C80A, C115A, C127A C30A, C64A, C76A, C80A, C94A, C115A C6A, C64A, C76A, C80A, C94A, C127A C6A, W62G, C64A, C76A, C80A, C94A, C127A

Additionally, for the mutants 1SS64-80-HEWL, 1SS76-94-HEWL and 1SS30-115-HEWL also the C6S instead of the C6A mutants were used. Both the C6A and C6S mutants are structurally and dynamically equivalent. Plasmids. DNA sequences of 4SS-HEWL, 0SS-HEWL and 0SSW62G-HEWL were available in pET11a plasmids.1 For 1SS64-80-HEWL, 1SS76-94-HEWL and 1SS6-127-HEWL the plasmid containing the 0SS-HEWL gene was used as template to reintroduce cysteine residues by two consecutive site directed mutageneses using the QuikChange® site directed mutagenesis kit (Stratagene, now Agilent Technologies, Santa Clara, US). For 1SS30-115-W62G-HEWL the plasmid containing 0SSW62G-HEWL was used in a similar fashion. 1SS30-115HEWL was then generated by reintroduction of tryptophan at position 62. 0SS-HEWL and 0SSW62G-HEWL DNA has the following sequence and the following sets of primer were used:

0SS-HEWL (5’3’): CATATGAAAG_TTTTTGGCCG_TGCTGAGTTA_GCGGCAGCA_ATGAAACGTC_ATGGTTTGGA_CAACTATCGC GGCTATAGTC_TGGGGAATTG_GGTTGCTGCG_GCCAAATTT_GAAAGCAATT_TCAATACCCA_AGCGACCAAC CGTAATACGG_ATGGCTCGAC_GGACTACGGT_ATTCTGCAG_ATCAACAGCC_GCTGGTGGGC_GAACGACGGT CGTACGCCGG_GCAGCCGTAA_TCTGGCAAAT_ATCCCAGCC_TCGGCGCTTC_TCAGTTCGGA_TATTACCGCA AGTGTTAACG_CGGCAAAAAA_GATCGTGAGC_GATGGCAAC_GGCATGAACG_CGTGGGTGGC_CTGGCGTAAC CGCGCCAAAG_GTACCGATGT_GCAGGCCTGG_ATTCGCGGG_GCTCGCCTGT_AAGGATCC

PAGE S2

0SSW62G-HEWL (5’3’): CATATGAAAG_TTTTTGGCCG_TGCTGAGTTA_GCGGCAGCA_ATGAAACGTC_ATGGTTTGGA_CAACTATCGC GGCTATAGTC_TGGGGAATTG_GGTTGCTGCG_GCCAAATTT_GAAAGCAATT_TCAATACCCA_AGCGACCAAC CGTAATACGG_ATGGCTCGAC_GGACTACGGT_ATTCTGCAG_ATCAACAGCC_GCGGGTGGGC_GAACGACGGT CGTACGCCGG_GCAGCCGTAA_TCTGGCAAAT_ATCCCAGCC_TCGGCGCTTC_TCAGTTCGGA_TATTACCGCA AGTGTTAACG_CGGCAAAAAA_GATCGTGAGC_GATGGCAAC_GGCATGAACG_CGTGGGTGGC_CTGGCGTAAC CGCGCCAAAG_GTACCGATGT_GCAGGCCTGG_ATTCGCGGG_GCTCGCCTGT_AAGGATCC

1SS64-80-HEWL: A64C_fwd A64C_rev A80C_fwd A80C_rev

5’-CAA 5’-GCG 5’-CTG 5’-GAA

CAG TAC GCA CTG

CCG GAC AAT AGA

CTG CGT ATC AGC

GTG CGT CCA GCC

GTG TGC TGC GAG

CAA ACC TCG CAT

CGA ACC GCG GGG

CGG AGC CTT ATA

TCG GGC CTC TTT

TAC TGT AGT GCC

GC-3’ TG-3’ TC-3’ AG-3’

1SS76-94-HEWL: A76C_fwd A76C_rev A94C_fwd A94C_rev

5’-GGC 5’-CGC 5’-TTA 5’-GCT

AGC CGA CCG CAC

CGT GGC CAA GAT

AAT TGG GTG CTT

CTG GAT TTA TTT

TGC ATT ACT TGC

AAT GCA GCG GCA

ATC CAG CAA GTT

CCA ATT AAA AAC

GCC ACG AGA ACT

TCG GCT TCG TGC

GCG-3’ GCC-3’ TGA GC-3’ GGT AA-3’

1SS6-127-HEWL: A6C_fwd A6C_rev A127C_fwd A127C_rev

5’-GAA 5’-GCT 5’-GCC 5’-GGA

AGT GCC TGG TCC

TTT GCT ATT TTA

TGG AAC CGC CAG

CCG TCA GGG GCG

TTG CAA TGT ACA

TGA CGG CGC CCC

GTT CCA CTG GCG

AGC AAA TAA AAT

GGC ACT GGA CCA

AGC-3’ TTC-3’ TCC-3’ GGC-3’

1SS30-115-W62G-HEWL: A30C_fwd 5’-CTG A30C_rev 5’-GCT A115C_fwd 5’-GCC A115C_rev 5’-GCA

GGG TTC TGG CAT

AAT AAA CGT CGG

TGG TTT AAC TAC

GTT GGC CGC CTT

TGT CGC TGC TGC

GCG ACA AAA AGC

GCC AAC GGT GGT

AAA CCA ACC TAC

TTT ATT GAT GCC

GAA CCC GTG AGG

1SS30-115-HEWL G62W_fwd G62W_rev

AGC-3’ CAG-3’ C-3’ C-3’

5’- GAT CAA CAG CCG CTG GTG GGC GAA CGA CGG-3’ 5’- CCG TCG TTC GCC CAC CAG CGG CTG TTG ATC-3’

Expression and purification of lysozyme mutants. Expression of recombinant lysozyme mutants was performed as described earlier.1 Lysozyme mutants were overexpressed in E. coli BL21(DE3) strain (Invitrogen, Carlsbad, US) using LB medium (10g/L tryptone, 5g/L yeast extract, 10g/L NaCl) or – for isotope labelling – the minimal medium M9 (42 mM di-sodiumhydrogenphosphat, 22 mM potassium-di-hydrogenphosphat, 8.5 mM sodium chloride, 0.1 mM calcium chloride, 2 mM magnesium sulfate, 0.5ml/L vitamin mix stock solution, 0.2ml/L trace elements stock solution, 1 g/L [15N]ammonium chloride, 10 g/L glucose) was used. Vitamin mix stock solution consists of 100mM thiamine, 100mM biotin, 100mM niacin and 10mM cobalamin in water. Trace element stock solution consists of 50mM FeCl3, 10mM MnCl2, 10mM ZnSO4, 2mM CoCl2, 2mM CuCl2, 2mM NiSO4, 2mM Na2MoO4, 2mM NaSeO3, 2mM H2BO3 and 5g/L EDTA as stabilizer in water. Cultures were induced at an OD600 = 0.8 with 2mM IPTG and allowed to grow for an addition 3h after induction. Cells were subsequently collected by low speed centrifugation and lysed by sonication in buffer containing 50mM Tris/HCl PAGE S3

pH 7.5, 25% (w/v) sucrose, 5mM EDTA. The insoluble fraction was washed twice with buffer containing 50mM Tris/HCl pH 7.5, 1% Triton X-100, 5mM EDTA. The insoluble fraction consisting of inclusion bodies was dissolved in buffer containing 50mM Tris/HCl pH 7.5, 50mM NaCl, 8M urea, 5mM EDTA, 0,1M DTT. After the insoluble fraction was removed by centrifugation, the supernatant was loaded onto a fast-flow CMSepharose column (GE Healthcare) equilibrated with buffer containing 50 mM Tris/HCl pH 7.5, 50 mM NaCl, 4 M urea, 1 mM EDTA, and 5 mM 2-mercaptoethanol. The column was eluted with a linear gradient between buffers containing 50 mM Tris/HCl pH 7.5, 50 mM NaCl, 4 M urea, 1 mM EDTA, and 5 mM 2mercaptoethanol and 50 mM Tris/HCl pH 7.5, 300 mM NaCl, 4 M urea, 1 mM EDTA, and 5 mM 2mercaptoethanol. Fractions containing lysozyme were merged and stored at -80°C for further purification. For 4SS-HEWL, the fractions obtained from CM-Sepharose purification were introduced into a refolding buffer containing 50 mM Tris/HCl pH 7.5, 50 mM NaCl, 5mM oxidized glutathione, 5mM reduced glutathione with a final concentration of 3µM. After 12h at 25°C the refolding mixture was concentrated by ultrafiltration and the insoluble fraction was removed by centrifugation. The soluble fraction was dialyzed against pH 7.0 water and freeze-dried for storage at -20°C. Monodispersity of the sample was checked by polyacralamide gelelectrophoresis and MALDI-TOF mass spectrometry. The yield of the purified folded 4SS-HEWL ranged from 3-5mg per liter medium. For 0SS-HEWL, the fractions obtained from CM-Sepharose purification were dialyzed against pH 2.0 water and freeze-dried. The samples were further purified with reverse-phase HPLC on a Waters µBondasphere C4 column (19x150 mm). Fractions containing 0SS-HEWL were pooled and freeze-dried for storage at -20°C. Monodispersity of the sample was checked by polyacralamide gelelectrophoresis and MALDI-TOF mass spectrometry. The yield of the purified 0SS-HEWL ranged from 8-12mg per liter medium. For 1SS-HEWL mutants, the fractions obtained from CM-Sepharose purification were introduced into an oxidizing buffer containing 100 mM Tris/Acetate pH 7.85, 2 µM CuCl2, at 25°C for 24 h at the protein concentration of 3 µM for air-oxidation as previously described.2 The solution was concentrated by ultrafiltration and the insoluble fraction was removed by centrifugation. The soluble fraction was dialyzed against pH 2.0 water and freeze-dried. The samples were further purified with reverse-phase HPLC on a Waters µBondasphere C4 column (19x150 mm). Fractions containing 1SS-HEWL mutants were pooled and freezedried for storage at -20°C. Monodispersity of the sample was checked by polyacralamide gelelectrophoresis and MALDI-TOF mass spectrometry. The yield of purified 1SS-HEWL mutants ranged from 2-4mg per liter medium.

Circular dichroism spectroscopy. Circular dichroism (CD) spectra were obtained using a Jasco J-810 spectropolarimeter. All measurements were performed at 20°C using a quartz cell with 0.2mm pathlength. Baseline corrected CD spectra were recorded at 0.5 nm increments between 180 and 250nm. Protein solutions were prepared in water pH 2.0 at a typical concentration of 20µM. Helical content was estimated according to PAGE S4

an approach by Rohl and Baldwin.3 The -helical content of a polypeptide can be calculated from the mean molar residue ellipticity at a wavelength of 222nm ([]222nm). The mean -helical content is then given by

(1)

where C and H are the ellipticities of the fully unfolded (random coil) and fully -helical polypeptide, respectively. Both vary with temperature and can be calculated from (2) (3)

where T ist he temperature in °C and N is the number of residues in the protein.

Small-angle X-ray scattering (SAXS). The SAXS measurements were recorded on the ID14-3 BioSAXS beamline at the European Synchrotron Radiation Facility (ESRF Grenoble, France). Sample-detector distance was 2.6 m, the X-ray wavelength used 0.931 Å (13.32 keV). 50 μL of each protein solution (as well as corresponding buffers) were loaded in a flow-through quartz capillary cell at 20 °C. Total exposure time was 100 s per sample. The 2D diffraction patterns were normalized to an absolute scale and azimuthally averaged to obtain the intensity profiles I(q), within BSxCuBE

(ESRF beamline data collection software). Solvent

contributions (buffer backgrounds collected before and after every protein sample) were averaged and subtracted from the associated protein sample using the program PRIMUS.4 The radii of gyration were extracted using the Ensemble Optimization Method (EOM).5 As ensemble pool of 10.000 structures generated with the flexible meccano approach were used for EOM. This pool of structures represents statistical coils derived from /-databases and is used to generate a sub-ensemble of structures fitting the SAXS curve by selection of adequate structures by the EOM algorithm.

Random flight model for branched polymers. In a polymer containing N units there is a reference unit that for simplification is the first unit of the polymer. Furthermore, the center of mass is given. The vector xi from the reference unit to unit i, vector ri from the center of mass to unit i and vector z from the reference unit to the center of mass. The index i ranges from 1 to N, latter indicating the total number of polymer segments. According to polymer theory, for linear unbranched polymers these are defined as (4)

PAGE S5

(5) (6)

For closed, cyclic polymers X2, Av and R2 are defined as: (7) (8) (9)

According to Zimm and Stockmayer,6 if several polymers are linked at one branch unit, as in the case of single disulfide bonds, X2 and Av can be calculated as the sums of each individual branch. ∑

(10)

∑

(11) (12)

The combination of equations (1), (2), (4), (5), (7)-(9) leads to

√

√(

)

(

)

(

)

(13)

Combination of equations (10) and (3) lead to √

√(

)

(

)

(

)

(14)

Ensemble calculations. The flexible meccano algorithm was utilized to generate 10.000 structures of 0SSHEWL, 1SS64-80-HEWL, 1SS76-94-HEWL, 1SS6-127-HEWL, 1SS30-115-HEWL, 1SS30-115W62G-HEWL each. In the case of single disulfide mutants the disulfide bond was incorporated as an additional constraint – the C/C PAGE S6

atom distance of the two cysteine residues is to be under 6Å. These ensembles were subsequently analyzed by HYDROPRO to yield radius of gyration (Rg) and hydrodynamic radius (Rh) distributions. Average Rh values are calculated as arithmetic average. Average Rg values were calculated with equation (15). √

∑

(15)

Resonance assignment and chemical shift analysis. All nuclear magnetic resonance (NMR) spectra were recorded on Bruker DRX600 MHz spectrometers and all experiments involving heteronuclear experiments were acquired on a uniformly

15

N-labeled sample. The data were processed using TOPSPIN 2.1 software (Bruker

BioSpin). For resonance assignment typically 3D-1H,15N-NOESY-HSQC and 3D-1H,15N-TOCSY-HSQC spectra were recorded and resonance assignment was carried out using the CARA software package. The resonance assignment was facilitated by the existence of assignments for 0SS-HEWL and 4SS-HEWL. For chemical shift analysis, proton chemical shifts were referenced to 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS).

15

N chemical shifts were calculated relative to DSS using the gyromagnetic ratios of

15

N and 1H.

Chemical shift deviations from random coil model were calculated according to Wishart et al.7,8

Backbone dynamics and relaxation. Relaxation analysis was carried out with data sets obtained from heteronuclear 1H-15N relaxation rates of backbone amides. For that,

15

N transverse (R2) and longitudinal (R1)

relaxation rate constants, rotating frame longitudinal relaxation rates (R1) and the heteronuclear 1H-15N-NOE (hetNOE) were measured. R2, R1 and R1 relaxation rates were measured as pseudo-three-dimensional spectra, hetNOE were recorded in an interleaved manner. For R2, R1, R1 and hetNOE the Bruker standard sequences were used. All spectra were recorded at 293K with a sample concentration of typically 300µM. For R 2 rates ten increments with delays ranging from 17ms to 374ms including three repeats were used. For R1 rates ten increments with delays ranging from 100ms to 2000ms including two repeats were used. For R1 rates ten increments with delays ranging from 20ms to 250ms including two repeats were used. Data was processes in Topspin 2.1 and exported to the Sparky software package (T. D. Goddard and D. G. Kneller, SPARKY 3, University of California, San Francisco) for relaxation analysis of R2, R1 and R1 data. The Sparky software package was also used to export intensities for hetNOE analysis. For the measurement of chemical exchange, CPMG relaxation experiments were performed as pseudo-three-dimensional experiments using the Bruker standard sequence. Typically, 18 different CPMG field strengths ranging from 25Hz to 750Hz were recorded with one additional reference spectrum without CPMG field. All spectra were recorded at 293K with a sample concentration of typically 300µM and were processes using Topspin 2.1 and exported to Sparky for resonance intensity extraction. The resulting effective relaxation rates R2eff were calculated as follows. (16) PAGE S7

Reduced spectral density analysis. Heteronuclear relaxation can be analysed by reduced spectral density mapping translating R2, R1 and hetNOE data into spectral densities.9 J(0), J(N) and J(0.87H) can be calculated by

[

]

(17) [

] [

]

where d² = (µ0hNH)²/(164r6) and c² = N22/3, r = 0.102nm,  = -160ppm and N=3.82×10-8 rad·s-1 at 14.1T. If the dynamic is limited to only a single motional mode, J() can be described by (18)

Residual dipolar couplings. Residual dipolar couplings (RDCs) were measured in stretched 7% polyacrylamide gels. For gel preparation, a gel preparation system from New Era Enterprises was used. Spectra of isotropically and anisotropically tumbling samples were measured at 293K and at a sample concentration of typically 300µM. A pseudo-three-dimensional spectrum was recorded in a J-modulated manner.10 RDCs were obtained by the subtraction of couplings isotropically and anisotropically tumbling samples. RDCs were normalized to 10Hz HDO splitting.

Diffusion-ordered spectroscopy. Translational diffusion was measured using diffusion-ordered spectroscopy (DOSY) in 100% D2O pH 2.0 (corrected for D2O) at 20°C and at a typical sample concentration of 300µM, 150µM and 75µM. Dioxane was used as internal diffusion standard in five-fold molar excess compared to the protein sample. For the measurement the standard Bruker sequence was used utilizing stimulated echo, longitudinal eddy delay (LED) and bipolar gradient pulses for diffusion. The gradient strength was varied from 2-95% in 32 linear steps. The number of scans typically varied from 16 to 128 scans depending on sample concentration. A diffusion time () of 150ms and a gradient pulse of 2.5ms was used. Data was processed and analysed using Topspin 2.1 (Bruker) and hydrodynamic radii were calculated by equation (19). (19) with respectively, and

, Ddioxane and Dprotein being the diffusion coefficients of dioxane and the sample protein, the hydrodynamic radius of the protein.

PAGE S8

SUPPORTING FIGURES AND TABLES

Figure S1: 1H-15N-HSQC spectrum of 300µM 0SS-HEWL in water pH 2.0 recorded at 293K on a Bruker DRX600MHz spectrometer.

PAGE S9

Figure S2: 1H-15N-HSQC spectrum of 300µM 4SS-HEWL in water pH 3.8 recorded at 293K on a Bruker DRX600MHz spectrometer.

PAGE S10

Figure S3: 1H-15N-HSQC spectrum of 300µM 1SS64-80-HEWL in water pH 2.0 recorded at 293K on a Bruker DRX600MHz spectrometer.

PAGE S11

Figure S4: 1H-15N-HSQC spectrum of 300µM 1SS76-94-HEWL in water pH 2.0 recorded at 293K on a Bruker DRX600MHz spectrometer.

PAGE S12

Figure S5: 1H-15N-HSQC spectrum of 300µM 1SS6-127-HEWL in water pH 2.0 recorded at 293K on a Bruker DRX600MHz spectrometer.

PAGE S13

Figure S6: 1H-15N-HSQC spectrum of 300µM 1SS30-115-HEWL in water pH 2.0 recorded at 293K on a Bruker DRX600MHz spectrometer.

PAGE S14

Figure S7: 1H-15N-HSQC spectrum of 300µM 1SS30-115W62G-HEWL in water pH 2.0 recorded at 293K on a Bruker DRX600MHz spectrometer.

PAGE S15

Figure S8: Compactness of lysozyme mutants. Panels A-F show the distributions of the radius of gyration (Rg) and the hydrodynamic radius (Rh) of (A) 0SS-HEWL, (B) 1SS64-80-HEWL, (C) 1SS76-94-HEWL, (D) 1SS6-127HEWL, (E) 1SS30-115-HEWL and (F) 1SS30-115W62G-HEWL. Distribution were compiled using the flexible meccano

11

algorithm to generate 10.000 structures, that were subsequently analysed by HYDROPRO

yielding Rg and Rh values. Average Rg and Rh values are indicated.

PAGE S16

12,13

Figure S9: Kratky and Porod-Debye plots of SAXS data obtained for A) 0SS-HEWL, (B) 1SS64-80-HEWL, (C) 1SS76-94-HEWL, (D) 1SS6-127-HEWL, (E) 1SS30-115-HEWL, (F) 1SS30-115W62G-HEWL and (G) 4SS-HEWL. Stationary points are indicated by *.

PAGE S17

Figure S10: Relaxation rate distributions for (A) 1SS64-80-HEWL, (B) 1SS76-94-HEWL, (C) 1SS6-127-HEWL, (D) 1SS30-115-HEWL and (E) 1SS30-115W62G-HEWL in red. For clarity, the relaxation rate distributions of 0SSHEWL (light gray) and 4SS-HEWL (dark grey) are shown mirrored.

PAGE S18

Figure S11: Spectral densities as a function of sequence for (A) 1SS64-80-HEWL, (B) 1SS76-94-HEWL, (C) 1SS6127

-HEWL, (D) 1SS30-115-HEWL and (E) 1SS30-115W62G-HEWL. For J(0) (left panel) and J(wN) (right panel)

also the correlations of R2/J(0) and R1/J(N) are shown, respectively.

PAGE S19

Figure S12: R2/R1 correlations of (A) 0SS-HEWL, (B) 1SS6-127-HEWL, (C) 1SS64-80-HEWL, (D) 1SS30-115HEWL, (E) 1SS76-94-HEWL and (F) 1SS30-115W62G-HEWL. Bidirectional error bars are given and a correlation coefficient of 1 is indicated by a line. Significant residues are indicated.

PAGE S20

Figure S13: CPMG experiments of 1SS76-94-HEWL, 1SS6-127-HEWL, 1SS30-115-HEWL and 1SS64-80-HEWL for five residues each.

PAGE S21

Table S1: NMR spectroscopic data of 1SS6-127-HEWL. R1. R2 and R1are fractional errors derived from fitting the exponential decay of intensity profiles in SPARKY. 1

HN

K1 V2 F3 G4 R5 C6 E7 L8 A9 A10 A11 M12 K13 R14 H15 G16 L17 D18 N19 Y20 R21 G22 Y23 S24 L25 G26 N27 W28 V29 A30 A31 A32 K33 F34 E35 S36 N37 F38 N39 T40 Q41 A42 T43 N44 R45 N46 T47 D48 G49 S50 T51 D52 Y53 G54 I55 L56 Q57 I58

15

N

H

1

H-15N RDC

R1

R1

R2

R2

R1

R1

[Hz]

[Hz]

[Hz]

1

[ppm]

[ppm]

[ppm]

[Hz]

[Hz]

[Hz]

[Hz]

8.707 8.167 8.433 8.324 8.276 8.559 — — — — 8.104 8.104 — 8.135 8.378 8.354 8.183 8.503 8.252 8.090 8.237 7.804 7.932 8.276 8.237 8.230 8.159 8.081 7.798 7.987 8.018 8.041 8.057 8.120 — — 8.276 8.050 8.308 8.104 8.316 8.268 8.081 8.354 8.316 8.487 8.165 8.495 8.378 8.167 8.198 8.378 8.120 8.269 7.846 8.198 8.253 8.073

125.638 124.983 125.261 110.992 121.248 119.412 — — — — 122.275 118.911 — 121.619 119.035 110.401 122.151 119.684 119.381 121.211 123.796 109.128 120.488 118.089 124.612 108.721 119.486 122.479 122.256 126.257 122.912 122.318 120.160 120.840 — — 120.612 120.760 120.612 115.015 122.621 125.373 113.207 121.477 121.929 120.142 114.631 121.496 110.112 116.185 116.246 121.681 121.372 110.233 120.327 125.292 121.805 121.990

— 4.223 4.654 3.945 4.261 4.654 — — — — 4.160 4.211 — 4.261 4.654 3.995 4.325 4.642 4.642 4.490 4.160 3.831 4.591 4.426 4.325 3.831 4.642 4.540 3.818 4.109 4.109 4.160 4.211 4.604 — — — 4.528 4.718 4.261 4.274 4.287 4.300 4.692 4.312 4.756 4.325 4.718 3.983 — — 4.692 4.426 3.881 4.147 4.312 4.312 4.033

-3.997 1.193 -3.099 -1.227 -2.380 -5.196 — — — — 0.173 -1.428 — -2.811 -3.263 -4.207 -4.380 -4.212 -2.654 -1.568 -2.939 -1.783 -2.061 -3.385 -2.635 -0.798 -2.707 -0.722 0.341 -1.395 -0.594 -0.151 -1.883 -0.798 — — -2.936 -2.371 -3.281 -3.226 -3.428 -2.009 -3.913 -5.182 -3.274 -2.311 -3.546 -2.304 -0.330 -1.918 -3.124 -2.392 -0.975 -0.420 -0.523 -1.631 -2.729 -0.824

1.013 1.423 1.470 1.527 1.520 1.601 — — — — 1.379 1.518 — 1.500 1.548 1.460 1.473 1.500 1.557 1.542 1.597 1.559 1.605 1.414 1.547 1.484 1.505 1.460 1.486 1.594 1.452 1.455 1.402 1.517 — — 1.541 1.518 1.559 1.515 1.539 1.520 1.400 1.526 1.532 1.549 1.488 1.559 1.459 1.436 1.497 1.573 1.606 1.622 1.581 1.491 1.644 1.487

0.009 0.023 0.017 0.012 0.033 0.112 — — — — 0.022 0.018 — 0.033 0.022 0.028 0.023 0.025 0.015 0.019 0.032 0.046 0.036 0.014 0.019 0.032 0.018 0.011 0.037 0.015 0.015 0.027 0.011 0.015 — — 0.027 0.023 0.013 0.022 0.013 0.009 0.015 0.014 0.012 0.015 0.015 0.013 0.015 0.009 0.003 0.018 0.032 0.009 0.009 0.021 0.030 0.014

2.162 3.978 4.552 5.656 7.128 17.449 — — — — 8.606 7.722 — 6.897 12.218 5.269 6.826 5.669 6.053 5.896 6.086 12.997 6.536 6.423 6.452 11.260 6.645 7.468 9.804 10.256 7.289 7.128 4.583 4.566 — — 4.822 6.238 4.292 3.775 4.885 3.529 3.205 3.623 3.754 3.532 3.122 3.217 2.972 3.466 3.982 4.241 5.408 5.189 5.848 6.892 7.479 7.310

0.028 2.036 0.025 0.065 3.818 0.064 0.123 4.314 0.055 0.095 5.322 0.053 0.149 6.868 0.113 0.837 14.422 0.786 — — — — — — — — — — — — 0.324 7.519 0.067 0.115 7.273 0.062 — — — 0.460 6.325 0.096 0.264 10.169 0.097 0.129 5.094 0.064 0.263 5.770 0.050 0.125 5.385 0.055 0.100 5.750 0.057 0.172 6.502 0.013 0.262 5.714 0.117 0.429 8.183 0.236 0.106 5.981 0.044 0.079 5.910 0.051 0.185 6.196 0.223 0.519 7.257 0.124 0.084 6.101 0.069 0.314 6.431 0.088 0.383 8.696 0.133 0.259 9.066 0.210 0.107 6.601 0.039 0.195 6.725 0.043 0.055 4.369 0.036 0.076 4.581 0.068 — — — — — — 0.053 4.444 0.130 0.075 6.083 0.043 0.053 4.170 0.077 0.072 3.736 0.076 0.215 4.486 0.105 0.104 3.514 0.034 0.052 3.306 0.033 0.128 3.605 0.049 0.324 3.293 0.030 0.056 3.410 0.026 0.103 3.157 0.012 0.274 3.337 0.033 0.048 2.934 0.079 0.145 3.459 0.028 0.075 3.445 0.047 0.138 4.191 0.037 0.191 5.299 0.038 0.063 4.975 0.029 0.072 5.441 0.068 0.141 6.046 0.072 0.163 6.916 0.067 0.802 6.196 0.019

PAGE S22

1

H-15N hetNOE

J(0)

J(0.87H)

J(N)

—

rad·ns-1

rad·ns-1

rad·ns-1

-0.943 0.042 -0.134 0.187 0.343 0.359 — — — — 0.136 0.239 — 0.152 0.047 0.068 0.238 0.100 0.279 0.125 0.162 0.224 0.331 0.161 0.238 0.309 0.243 0.235 0.174 0.332 0.181 0.253 -0.058 0.112 — — 0.192 0.113 0.123 0.064 0.107 0.066 -0.043 -0.040 0.011 0.077 -0.009 0.026 -0.044 0.082 0.093 0.047 0.139 0.248 0.246 0.106 0.280 0.066

0.440 0.900 1.051 1.358 1.776 4.663 — — — — 2.208 1.941 — 1.710 3.195 1.257 1.696 1.364 1.467 1.422 1.468 3.416 1.597 1.590 1.580 2.941 1.640 1.878 2.529 2.643 1.828 1.784 1.071 1.052 — — 1.122 1.522 0.969 0.829 1.138 0.759 0.685 0.783 0.820 0.756 0.649 0.665 0.610 0.755 0.891 0.951 1.276 1.214 1.405 1.709 1.855 1.827

0.031 0.021 0.026 0.019 0.016 0.016 — — — — 0.019 0.018 — 0.020 0.023 0.021 0.018 0.021 0.018 0.021 0.021 0.019 0.017 0.019 0.018 0.016 0.018 0.017 0.019 0.017 0.019 0.017 0.023 0.021 — — 0.019 0.021 0.021 0.022 0.021 0.022 0.023 0.025 0.024 0.022 0.023 0.024 0.024 0.021 0.021 0.023 0.022 0.019 0.019 0.021 0.018 0.022

0.143 0.234 0.236 0.257 0.261 0.275 — — — — 0.230 0.257 — 0.251 0.255 0.241 0.249 0.249 0.265 0.257 0.267 0.263 0.275 0.237 0.262 0.254 0.255 0.247 0.249 0.273 0.244 0.247 0.227 0.252 — — 0.259 0.252 0.259 0.250 0.256 0.251 0.227 0.248 0.251 0.256 0.243 0.256 0.237 0.238 0.248 0.259 0.268 0.275 0.268 0.248 0.280 0.245

1

HN

15

N

H

1

H-15N RDC

R1

R1

R2

R2

R1

R1

[Hz]

[Hz]

[Hz]

[Hz]

1

1

H-15N hetNOE

J(0)

J(0.87H)

J(N)

—

rad·ns-1

rad·ns-1

rad·ns-1

-0.042 0.147 0.099 0.492 0.330 0.257 0.227 0.174 0.151 0.239 0.211 — -0.006 0.012 0.024 -0.112 0.072 0.135 0.119 0.043 — 0.066 0.175 0.233 0.051 0.005 0.052 — -0.066 -0.035 0.024 0.095 0.020 -0.077 — — -0.080 0.182 -0.071 -0.009 -0.053 -0.234 0.066 -0.142 -0.088 -0.037 0.150 0.114 0.269 0.207 0.307 0.326 0.370 — 0.374 0.386 0.389 0.186

2.201 1.514 1.165 1.551 1.856 1.459 1.202 1.076 1.343 0.923 0.915 — 0.699 0.754 0.857 0.787 0.819 0.944 0.975 0.932 — 1.016 1.134 1.063 1.108 1.186 1.094 — 0.784 0.986 1.155 1.235 0.872 0.938 — — 1.098 1.575 1.005 1.101 1.350 1.023 0.876 0.687 0.824 0.801 1.142 1.399 1.547 1.880 2.132 2.729 2.364 — 2.364 1.807 1.659 1.613

0.026 0.019 0.021 0.013 0.019 0.020 0.019 0.021 0.021 0.019 0.019 — 0.023 0.023 0.023 0.026 0.021 0.020 0.020 0.021 — 0.022 0.019 0.018 0.022 0.024 0.022 — 0.025 0.024 0.023 0.021 0.021 0.023 — — 0.025 0.019 0.024 0.023 0.024 0.028 0.020 0.025 0.024 0.023 0.020 0.021 0.017 0.019 0.019 0.018 0.016 — 0.016 0.014 0.015 0.018

0.257 0.243 0.243 0.289 0.311 0.293 0.272 0.271 0.269 0.275 0.260 — 0.242 0.246 0.245 0.242 0.244 0.251 0.243 0.237 — 0.247 0.249 0.253 0.245 0.250 0.241 — 0.246 0.242 0.247 0.249 0.223 0.225 — — 0.238 0.253 0.230 0.240 0.240 0.228 0.232 0.227 0.233 0.233 0.257 0.252 0.258 0.252 0.293 0.288 0.278 — 0.280 0.261 0.276 0.242

[ppm]

[ppm]

[ppm]

[Hz]

[Hz]

[Hz]

N59 S60 R61 W62 W63 A64 N65 D66 G67 R68 T69 P70 G71 S72 R73 N74 L75 A76 N77 I78 P79 A80 S81 A82 L83 L84 S85 S86 D87 I88 T89 A90 S91 V92 N93 A94 A95 K96 K97 I98 V99 S100 D101 G102 N103 G104 M105 N106 A107 W108 V109 A110 W111 R112 N113 R114 A115 K116

8.308 8.222 8.214 7.799 7.376 7.713 8.112 8.330 8.307 7.971 8.206 — 8.480 8.104 8.401 8.409 8.237 8.174 8.245 7.971 — 8.480 8.229 8.159 7.909 7.987 8.183 8.229 8.378 8.065 8.127 8.221 8.229 8.159 — — 8.135 8.018 8.112 8.151 8.268 8.401 8.543 8.403 8.347 8.472 8.221 8.386 8.198 8.034 7.853 7.994 7.931 — 8.073 7.869 8.120 8.151

122.170 116.964 122.745 121.211 121.043 124.612 117.699 119.159 109.004 120.513 117.520 — 110.032 116.141 123.196 120.160 123.654 123.913 117.749 123.221 — 125.107 114.011 126.257 120.673 122.219 116.370 117.787 121.422 121.867 118.256 126.479 115.875 122.646 — — 122.708 119.771 122.788 123.363 125.267 120.018 121.966 109.924 119.258 109.911 120.593 119.752 124.346 120.284 121.329 124.655 119.919 — 118.478 121.459 124.717 120.779

4.705 4.300 4.008 4.490 4.464 4.160 4.642 4.642 3.881 4.376 4.528 — 3.945 4.388 4.338 4.642 4.261 4.261 4.642 4.363 — 4.160 4.211 4.300 4.287 4.287 — — 4.743 4.211 4.211 4.274 4.338 4.021 — — — — — 4.274 4.160 4.477 4.756 3.932 4.705 3.945 4.426 4.591 4.211 4.490 3.704 4.160 4.591 — 4.553 4.211 4.211 4.223

-2.997 -3.466 -3.923 6.479 4.442 4.392 -2.278 -1.193 -0.140 -2.190 -3.018 — -2.675 -2.275 -4.454 -4.538 -3.771 -1.735 -2.686 -7.615 — -4.230 -2.474 0.739 -2.181 -2.417 -1.020 — -1.907 0.720 -1.414 0.237 -0.459 -0.096 — — -4.500 -2.252 0.653 -6.915 -2.216 -4.431 -5.918 1.852 -0.853 -0.157 1.338 2.807 2.038 1.082 7.081 5.492 4.977 — 5.272 -1.582 -0.296 -1.572

1.583 1.454 1.462 1.651 1.813 1.728 1.611 1.615 1.608 1.624 1.540 — 1.484 1.501 1.491 1.504 1.477 1.503 1.458 1.438 — 1.498 1.486 1.499 1.485 1.529 1.460 — 1.517 1.489 1.506 1.500 1.358 1.391 — — 1.472 1.506 1.422 1.470 1.478 1.441 1.406 1.419 1.441 1.432 1.540 1.515 1.518 1.496 1.715 1.679 1.613 — 1.625 1.512 1.600 1.440

0.028 8.703 0.630 7.138 0.112 0.027 6.173 0.161 6.013 0.061 0.012 4.941 0.073 4.623 0.038 0.025 6.386 0.192 6.423 0.044 0.028 7.570 0.122 7.446 0.080 0.037 6.116 0.226 6.227 0.179 0.005 5.141 0.060 4.847 0.022 0.019 4.699 0.097 4.523 0.040 0.010 5.647 0.905 4.050 0.031 0.018 4.155 0.061 4.072 0.035 0.014 4.083 0.045 3.802 0.019 — — — — — 0.024 3.299 0.079 3.204 0.080 0.008 3.501 0.024 3.421 0.037 0.020 3.863 0.144 3.854 0.047 0.011 3.630 0.025 3.535 0.024 0.019 3.718 0.071 3.755 0.028 0.016 4.172 0.107 4.008 0.038 0.010 4.261 0.040 3.954 0.023 0.017 4.100 0.056 3.994 0.053 — — — — — 0.011 4.429 0.058 4.354 0.035 0.032 4.838 0.147 4.785 0.060 0.014 4.585 0.120 4.427 0.066 0.016 4.751 0.065 4.382 0.265 0.016 5.058 0.112 5.056 0.044 0.005 4.688 0.071 4.460 0.036 — — — — — 0.017 3.623 0.160 3.792 0.020 0.012 4.327 0.106 4.511 0.053 0.022 4.933 0.046 4.662 0.031 0.018 5.208 0.458 4.382 0.196 0.006 3.843 0.036 3.808 0.035 0.010 4.103 0.051 4.179 0.036 — — — — — — — — — — 0.016 4.719 0.093 4.399 0.054 0.044 6.418 0.073 5.988 0.025 0.008 4.359 0.090 4.008 0.036 0.027 4.721 0.099 4.486 0.023 0.026 5.618 0.221 5.084 0.146 0.018 4.444 0.042 4.139 0.054 0.021 3.881 0.319 3.635 0.053 0.018 3.229 0.039 3.180 0.141 0.011 3.726 0.030 3.569 0.032 0.019 3.635 0.046 3.712 0.115 0.023 4.895 0.059 4.728 0.031 0.033 5.801 0.120 5.593 0.030 0.025 6.317 0.136 6.028 0.087 0.042 7.496 0.058 6.949 0.051 0.027 8.503 0.187 8.389 0.137 0.060 10.607 0.410 10.150 0.340 0.036 9.268 0.121 8.636 0.074 — — — — — 0.037 9.276 0.076 8.361 0.094 0.032 7.231 0.448 6.897 0.142 0.024 6.752 0.157 6.443 0.091 0.016 6.515 0.082 6.293 0.078

G117

8.363

110.300

3.995

-4.470

1.429 0.006

4.864

0.069

4.531

0.085

0.070

1.148

0.021

0.236

T118

8.041

113.989

4.338

-3.959

1.395 0.017

5.621

0.103

5.628

0.044

0.168

1.368

0.018

0.234

PAGE S23

1

HN

D119 V120 Q121 A122 W123 I124 R125 G126 C127 R128 L129

15

N

H

1

H-15N RDC

R1

R1

R2

R2

R1

R1

[Hz]

[Hz]

[Hz]

1

[ppm]

[ppm]

[ppm]

[Hz]

[Hz]

[Hz]

8.526 8.081 8.065 — — — 8.042 8.307 — — —

122.275 121.576 123.975 — — — 123.697 109.628 — — —

4.756 3.881 4.147 — — — — 3.932 — — —

-5.200 -1.820 -1.375 — — — -2.988 -0.997 — — —

1.419 1.482 1.553 — — — 1.675 1.712 — — —

0.039 9.814 3.689 5.382 0.019 5.790 0.231 6.270 0.057 10.889 0.414 8.921 — — — — — — — — — — — — 0.065 11.766 0.413 9.709 0.083 10.496 2.049 12.566 — — — — — — — — — — — —

PAGE S24

1

H-15N hetNOE

J(0)

J(0.87H)

J(N)

[Hz]

—

rad·ns-1

rad·ns-1

rad·ns-1

0.078 0.042 0.176 — — — 0.100 0.827 — — —

0.274 0.146 0.431 — — — 0.675 0.355 — — —

2.544 1.402 2.829 — — — 3.063 2.694 — — —

0.016 0.020 0.014 — — — 0.008

0.241 0.247 0.270 — — — 0.301 0.295 — — —

— — —

Table S2: NMR spectroscopic data of 1SS30-115-HEWL. R1. R2 and R1are fractional errors derived from fitting the exponential decay of intensity profiles in SPARKY. 1

HN

K1 V2 F3 G4 R5 S6 E7 L8 A9 A10 A11 M12 K13 R14 H15 G16 L17 D18 N19 Y20 R21 G22 Y23 S24 L25 G26 N27 W28 V29 C30 A31 A32 K33 F34 E35 S36 N37 F38 N39 T40 Q41 A42 T43 N44 R45 N46 T47 D48 G49 S50 T51 D52 Y53 G54 I55 L56 Q57 I58

15

N

H

1

H-15N RDC

R1

R1

R2

R2

R1

R1

[Hz]

[Hz]

[Hz]

[Hz]

1

[ppm]

[ppm]

[ppm]

[Hz]

[Hz]

[Hz]

8.711 8.264 8.537 8.372 8.312 8.512 8.413 8.187 8.227 8.146 8.100 8.132 — 8.194 8.509 8.456 8.234 8.537 8.316 8.113 8.235 7.747 7.977 8.290 8.223 8.095 8.169 — 7.915 — — — 8.086 8.017 8.018 8.194 8.250 8.072 8.277 8.099 8.312 8.263 8.078 8.360 8.324 8.490 8.168 8.491 8.381 8.170 8.203 8.366 8.128 8.264 7.848 8.194 8.256 8.087

125.588 123.300 125.926 111.441 121.486 117.651 123.493 123.588 124.647 122.950 122.714 119.318 — 122.006 119.938 110.704 122.261 120.214 119.901 121.379 124.170 109.290 120.706 118.082 124.784 108.886 119.200 — 122.967 — — — 119.318 120.896 121.773 116.733 120.408 120.605 120.487 114.918 122.517 125.294 113.102 121.379 121.849 120.059 114.548 121.440 110.037 116.130 116.186 121.655 121.328 110.114 120.266 125.201 121.741 122.004

4.413 4.060 4.645 3.915 4.300 4.374 4.337 4.249 4.199 4.161 4.199 4.337 — 4.249 4.701 3.940 4.337 4.651 4.651 4.475 4.199 3.804 4.610 4.425 4.199 3.754 4.600 — 3.973 — — — 4.185 4.425 4.199 4.286 4.600 4.513 4.688 4.261 4.286 4.286 4.286 4.701 4.286 4.739 4.337 4.701 3.977 4.475 4.275 4.663 4.475 3.883 4.140 4.300 4.286 4.005

-6.807 -6.392 -4.255 — 3.429 — — — — 2.449 -3.043 1.603 — -5.128 -5.112 — -5.378 -8.316 -6.230 -2.288 -2.157 -3.536 — -3.960 -2.711 -1.366 -1.964 — — — — — -1.966 0.679 -5.310 -3.539 -2.289 -1.078 -4.273 -5.248 -5.109 -3.092 -6.333 -3.335 -3.808 -4.203 -6.327 -3.669 -1.528 -3.445 -2.996 -4.721 -0.406 -1.051 -0.385 -2.831 -4.633 -0.252

0.868 1.116 1.242 1.231 — 1.520 — — — — 1.470 1.449 — 1.548 1.494 1.345 1.456 1.427 1.454 1.521 1.535 1.486 1.567 1.413 1.509 1.504 1.557 — 1.597 — — — 1.392 1.474 1.388 1.422 1.445 1.486 1.535 1.480 1.553 1.520 1.448 1.510 1.514 1.626 1.489 1.640 1.501 1.469 1.469 1.582 1.576 1.570 1.570 1.562 1.392 1.511

0.005 1.335 0.108 1.115 0.013 0.009 1.410 0.021 1.665 0.014 0.014 1.582 0.138 1.722 0.030 0.009 1.721 0.067 1.931 0.032 — 2.516 0.065 2.407 0.035 0.024 2.854 0.057 2.585 0.019 — 2.639 0.066 2.758 0.030 — — — — — — — — — — — — — — — 0.014 3.551 0.192 — — 0.014 3.540 0.024 3.765 0.024 — — — — — 0.026 3.902 0.154 3.539 0.040 0.038 4.134 0.070 3.521 0.036 0.010 3.004 0.032 3.084 0.064 0.019 3.902 0.160 3.484 0.073 0.011 3.625 0.040 3.573 0.020 0.027 4.184 0.088 3.940 0.047 0.022 5.203 0.093 5.179 0.058 0.018 5.851 0.122 5.534 0.052 0.020 5.995 0.195 5.609 0.037 0.051 — — 5.865 0.021 0.028 7.764 0.188 5.757 0.048 0.018 — — — — 0.071 — — 10.675 0.228 0.159 — — 9.174 0.295 — — — — — 0.028 — — 9.276 0.364 — — — — — — — — — — — — — — — 0.038 9.506 0.548 9.533 0.072 0.040 11.533 0.368 10.342 0.193 0.059 — — 9.606 0.171 0.034 8.396 0.126 7.710 0.056 0.013 6.365 0.085 — — 0.015 6.748 0.059 6.667 0.093 0.020 6.223 0.088 6.113 0.036 0.047 4.857 0.090 4.988 0.074 0.021 5.099 0.101 5.277 0.092 0.017 4.299 0.094 4.560 0.064 0.032 3.827 0.061 4.322 0.064 0.014 5.356 0.051 4.931 0.038 0.018 4.252 0.016 4.212 0.044 0.018 4.134 0.085 3.989 0.024 0.012 3.458 0.084 3.870 0.056 0.037 4.078 0.039 3.923 0.023 0.015 3.498 0.073 3.668 0.078 0.014 4.325 0.023 4.181 0.057 0.017 4.325 0.025 4.533 0.030 0.015 5.417 0.069 4.836 0.055 0.039 5.203 0.058 — — 0.041 6.494 0.180 6.301 0.060 0.049 7.205 0.152 6.901 0.054 0.034 6.685 0.378 6.854 0.195 0.006 — — 8.361 0.094 0.041 6.743 0.167 6.378 0.109

PAGE S25

1

H-15N hetNOE

J(0)

J(0.87H)

J(N)

—

rad·ns-1

rad·ns-1

rad·ns-1

-1.412 -0.830 -0.752 -0.640 — 0.076 — — — — -0.050 -0.084 — -0.036 -0.108 -0.185 0.035 -0.020 0.060 0.065 0.214 -0.014 0.472 0.276 0.089 0.248 — — 0.284 — — — 0.324 0.331 0.312 0.170 -0.047 0.269 0.360 0.183 0.294 0.157 0.034 0.091 0.123 0.129 0.087 0.121 0.044 0.189 0.189 0.151 0.227 0.371 0.435 0.329 -0.200 0.135

0.226 0.213 0.242 0.284 — 0.570 — — — — 0.771 0.771 — 0.858 0.930 0.634 0.873 0.799 0.954 1.229 1.412 1.456 — 1.969 — — — — — — — — 2.462 3.020 — 2.144 1.566 1.673 1.520 1.141 1.200 0.978 0.854 1.275 0.965 0.915 0.745 0.897 0.753 0.993 0.993 1.282 1.224 1.591 1.792 1.645 — 1.665

0.033 0.032 0.034 0.031 — 0.022 — — — — 0.024 0.024 — 0.025 0.026 0.025 0.022 0.023 0.021 0.022 0.019 0.024 — 0.016 — — — — — — — — 0.015 0.015 — 0.018 0.024 0.017 0.015 0.019 0.017 0.020 0.022 0.021 0.021 0.022 0.021 0.022 0.022 0.019 0.019 0.021 0.019 0.015 0.014 0.016 — 0.020

0.113 0.160 0.181 0.183 — 0.251 — — — — 0.239 0.234 — 0.252 0.241 0.214 0.239 0.233 0.240 0.251 0.259 0.242 — 0.240 — — — — — — — — 0.238 0.253 — 0.238 0.235 0.252 0.264 0.249 0.265 0.254 0.238 0.250 0.252 0.271 0.247 0.273 0.247 0.247 0.247 0.264 0.266 0.271 0.273 0.268 — 0.252

1

HN

N59 S60 R61 W62 W63 A64 N65 D66 G67 R68 T69 P70 G71 S72 R73 N74 L75 A76 N77 I78 P79 A80 S81 A82 L83 L84 S85 S86 D87 I88 T89 A90 S91 V92 N93 A94 A95 K96 K97 I98 V99 S100 D101 G102 N103 G104 M105 N106 A107 W108 V109 A110 W111 R112 N113 R114 C115 K116 G117 T118

15

N

H

1

H-15N RDC

R1

R1

R2

R2

R1

R1

1

1

H-15N hetNOE

J(0)

J(0.87H)

J(N)

[ppm]

[ppm]

[ppm]

[Hz]

[Hz]

[Hz]

[Hz]

[Hz]

[Hz]

[Hz]

—

rad·ns-1

rad·ns-1

rad·ns-1

8.297 8.217 8.227 7.801 7.383 7.716 8.114 8.325 8.305 7.971 8.210 — 8.489 8.102 8.409 8.414 8.243 8.176 8.247 7.968 — 8.488 8.236 8.152 7.895 7.985 8.188 8.229 8.384 8.072 8.127 8.216 8.223 8.168 — — 8.114 8.046 8.106 8.128 8.263 8.393 8.537 8.408 8.360 8.489 — 8.399 8.256 8.052 — — 7.965 — 8.024 7.780 8.066 8.037 8.324

122.104 116.939 122.692 121.135 120.912 124.507 117.633 119.121 108.900 120.405 117.422 — 110.017 116.070 123.126 120.069 123.570 123.772 117.615 123.146 — 125.040 113.884 126.167 120.543 122.090 116.246 117.752 121.485 121.835 118.138 126.263 115.815 122.649 — — 122.590 119.882 122.405 123.135 125.037 119.900 121.913 109.843 119.238 109.863 — 119.606 124.372 120.366 — — 120.256 — 117.494 120.216 118.046 122.394 110.232

4.711 4.231 4.002 4.475 4.425 4.161 4.600 4.618 3.868 4.387 4.563 — 3.956 4.374 4.286 4.651 4.286 4.249 4.651 4.362 — 4.179 4.261 4.286 4.299 4.296 4.367 4.416 4.688 4.148 4.224 4.286 4.374 4.023 — — 4.249 4.199 4.249 4.111 4.123 4.462 4.776 3.928 4.726 3.953 — 4.563 4.186 4.387 — — 4.574 — 4.475 4.199 4.575 4.249 3.953

-3.990 -4.304 -2.105 6.098 6.048 3.540 -4.157 -3.349 -0.821 -2.838 -5.689 — -6.685 -4.131 -6.097 -6.288 -5.867 -0.988 -3.465 — — -0.257 -5.047 0.559 -0.679 -3.814 -0.872 -2.552 -1.527 -0.186 -0.421 0.996 -2.286 -2.022 — — — -1.242 -0.306 -3.595 -7.258 -5.190 -7.281 1.623 -3.688 -2.512 — 3.305 1.422 — — 0.907 — — — — 2.014 -3.948

1.538 1.466 1.613 1.607 1.948 1.691 1.512 1.586 1.582 1.530 1.498 — 1.493 1.489 1.547 1.572 1.521 1.485 1.445 1.480 — 1.454 1.463 1.605 1.390 1.456 1.399 1.457 1.460 1.459 1.493 1.463 1.346 1.448 — — — 1.436 1.497 1.433 1.457 1.371 1.432 1.426 1.445 1.422 — 1.463 1.401 1.374 — — — — 1.288 1.443 — — 1.386

0.040 0.026 0.034 0.063 0.159 0.057 0.018 0.032 0.027 0.022 0.019 — 0.017 0.018 0.019 0.017 0.022 0.019 0.008 0.014 — 0.018 0.018 0.044 0.033 0.025 0.026 0.025 0.022 0.018 0.023 0.016 0.021 0.009 — — — 0.018 0.019 0.008 0.030 0.019 0.014 0.032 0.007 0.029 — 0.037 0.038 0.033 — — — — 0.066 0.084 — — 0.036

8.718 8.621 7.770 9.579 11.858 8.217 5.956 5.374 6.017 5.549 4.854 — 3.587 4.021 3.888 4.250 4.405 4.838 5.053 5.105 — 5.540 6.636 5.348 5.910 — 4.325 5.214 5.356 5.794 6.278 6.031 4.810 5.053 — — — 6.124 — 7.639 8.857 5.963 4.653 3.934 4.598 4.907 — 8.562 9.980 11.827 — — — — 13.208 11.798 — 11.905 8.945

0.420 0.252 0.226 0.347 0.219 0.332 0.050 0.036 0.391 0.040 0.091 — 0.055 0.042 0.094 0.053 0.085 0.083 0.044 0.084 — 0.176 0.170 0.154 0.070 — 0.041 0.076 0.036 0.051 0.049 0.118 0.028 0.110 — — — 0.108 — 0.469 0.101 0.071 0.060 0.080 0.111 0.130 — 0.115 0.121 0.260 — — — — 0.539 0.586 — 0.371 0.267

7.358 7.380 7.003 8.045 9.833 7.593 5.643 5.115 5.187 — 4.583 — 4.246 4.188 3.954 4.155 4.427 4.924 4.577 5.977 — 5.322 6.090 5.767 5.931 6.863 — 4.799 — 5.734 6.050 5.599 4.735 4.953 — — — 5.593 4.077 7.153 7.468 5.596 4.859 4.259 4.684 4.246 — 7.770 9.597 10.707 — — — — 12.771 12.127 — 10.802 8.764

0.105 0.143 0.089 0.079 0.431 0.187 0.052 0.038 0.075 — 0.089 — 0.014 0.063 0.088 0.047 0.036 0.068 0.017 0.188 — 0.050 0.035 0.045 0.086 0.122 — 0.045 — 0.084 0.082 0.063 0.035 0.032 — — — 0.042 0.037 0.236 0.144 0.072 0.057 0.089 0.039 0.024 — 0.176 0.222 0.093 — — — — 0.357 0.432 — 0.089 0.230

0.391 0.383 0.501 0.248 0.352 0.365 0.324 0.222 0.177 0.271 0.202 — 0.092 0.089 -0.017 0.029 0.109 -0.009 0.121 0.150 — -0.187 0.291 0.246 0.117 0.212 0.098 0.220 0.019 0.073 0.085 0.139 0.155 0.081 — — — -0.002 -0.069 0.039 0.054 -0.014 0.137 0.022 0.107 0.146 — 0.299 0.477 0.456 — — — — 0.619 0.473 — 0.142 0.408

2.221 2.204 1.935 2.449 3.041 2.057 1.447 1.271 1.451 1.330 1.138 — 0.780 0.903 0.855 0.953 1.006 1.131 1.200 1.210 — 1.330 1.645 1.261 1.449 — 1.002 1.245 1.281 1.405 1.536 1.473 1.147 1.199 — — — 1.500 — 1.927 2.266 1.464 1.090 0.887 1.072 1.163 — 2.186 2.597 3.120 — — — — 3.513 3.102 — — 2.307

0.015 0.014 0.025 0.019 0.020 0.017 0.016 0.019 0.020 0.017 0.019 — 0.021 0.021 0.025 0.024 0.021 0.023 0.020 0.020 — 0.027 0.016 0.019 0.019 — 0.020 0.018 0.022 0.021 0.021 0.020 0.018 0.021 — — — 0.022 — 0.021 0.021 0.022 0.019 0.022 0.020 0.019 — 0.016 0.011 0.012 — — — — 0.020 0.012 — — 0.013

0.266 0.253 0.264 0.272 0.335 0.291 0.259 0.268 0.265 0.260 0.252 — 0.247 0.247 0.252 0.258 0.253 0.242 0.240 0.247 — 0.231 0.249 0.272 0.231 — 0.232 0.246 0.239 0.241 0.247 0.244 0.225 0.240 — — — 0.235 — 0.236 0.240 0.224 0.239 0.234 0.240 0.237 — 0.250 0.245 0.240 — — — — 0.211 0.252 — — 0.240

8.024

113.944

4.286

-4.615

1.404 0.044

9.728

0.126

8.636

0.125

0.359

2.524

0.014

0.242

PAGE S26

1

HN

D119 V120 Q121 A122 W123 I124 R125 G126 A127 R128 L129

15

N

H

1

H-15N RDC

R1

R1

R2

R2

R1

R1

1

1

H-15N hetNOE

J(0)

J(0.87H)

J(N)

[ppm]

[ppm]

[ppm]

[Hz]

[Hz]

[Hz]

[Hz]

[Hz]

[Hz]

[Hz]

—

rad·ns-1

rad·ns-1

rad·ns-1

8.495 8.081 8.333 — — 7.916 8.161 8.285 8.032 8.258 8.326

122.027 121.220 123.162 — — 123.064 124.792 110.463 124.184 120.800 125.512

4.739 3.928 4.161 — — 4.097 4.211 3.891 4.261 4.249 4.337

-5.555 0.168 -4.703 — — — 0.393 -4.200 -0.867 -3.944 -4.055

1.507 1.486 1.437 — — — — 1.383 1.441 1.282 1.139

0.050 0.051 0.028 — — — — 0.022 0.024 0.007 0.010

8.354 — — — — — — 6.192 3.939 4.688 3.097

0.141 — — — — — — 0.096 0.157 0.033 0.033

7.273 9.091 8.772 — — — — 5.331 4.346 4.838 2.900

0.086 0.194 0.239 — — — — 0.062 0.051 0.026 0.050

0.259 0.223 0.434 — — — — 0.044 -0.056 -0.195 -0.566

2.121 — — — — — — 1.528 0.884 1.117 0.687

0.017 — — — — — — 0.021 0.024 0.024 0.028

0.256 — — — — — — 0.228 0.234 0.204 0.171

PAGE S27

Table S3: NMR spectroscopic data of 1SS30-115W62G-HEWL. R1. R2 and R1are fractional errors derived from fitting the exponential decay of intensity profiles in SPARKY. 1

HN

K1 V2 F3 G4 R5 A6 E7 L8 A9 A10 A11 M12 K13 R14 H15 G16 L17 D18 N19 Y20 R21 G22 Y23 S24 L25 G26 N27 W28 V29 C30 A31 A32 K33 F34 E35 S36 N37 F38 N39 T40 Q41 A42 T43 N44 R45 N46 T47 D48 G49 S50 T51 D52 Y53 G54 I55 L56 Q57 I58

15

N

H

1

H-15N RDC

R1

R1

R2

R2

R1

R1

[Hz]

[Hz]

[Hz]

[Hz]

1

[ppm]

[ppm]

[ppm]

[Hz]

[Hz]

[Hz]

8.709 8.252 8.511 8.370 8.307 8.466 8.245 8.189 — — 8.088 8.112 8.087 8.174 8.496 8.448 8.227 8.538 8.315 8.116 8.237 7.760 7.963 8.290 8.221 8.104 8.174 8.010 7.928 — — — 8.096 8.026 8.017 8.206 8.290 8.094 8.276 8.104 8.322 8.280 8.094 8.378 8.331 8.505 8.174 8.503 8.394 8.182 8.206 8.385 8.125 8.276 7.853 8.220 8.293 8.112

125.611 123.265 125.766 111.387 121.782 125.080 120.294 123.891 — — 122.640 119.204 122.485 121.937 119.907 110.684 122.330 120.212 119.907 121.467 124.201 109.354 120.460 118.186 124.666 108.965 119.240 122.170 123.347 — — — 119.437 120.997 121.937 116.858 120.609 120.764 120.532 114.983 122.640 125.451 113.262 121.467 121.937 120.184 114.668 121.467 110.136 116.161 116.233 121.705 121.433 110.296 120.294 125.611 122.092 122.247

4.381 4.109 4.652 3.943 4.215 4.215 4.274 4.274 — — 4.221 4.380 4.274 4.274 4.709 3.953 4.352 4.659 4.656 4.493 4.215 3.836 4.586 4.433 4.270 3.775 4.649 4.655 4.109 — — — 4.215 4.437 4.215 4.269 4.545 4.599 4.713 4.275 4.281 4.329 4.322 4.715 4.327 4.764 4.322 4.710 3.998 4.489 4.288 4.710 — 3.884 4.166 4.317 4.327 4.052

-5.219 -6.940 -2.869 -0.729 -0.242 -3.308 0.819 -0.160 — — 2.590 0.400 -2.315 -3.525 -4.813 -3.772 -3.712 -4.828 -4.625 -1.156 -1.619 -2.329 -1.654 -3.702 0.118 -1.040 -1.260 -2.283 -0.817 — — — -2.396 -5.047 -4.049 -3.952 -3.864 -1.092 -3.617 -3.908 -4.391 -2.038 -5.131 -6.091 -3.828 -2.915 -4.278 -2.634 -0.629 -2.418 -3.859 -2.469 -0.023 -0.973 -1.784 -4.241 -4.989 -3.404

0.849 1.079 1.188 1.248 1.361 1.439 1.408 1.464 — — 1.467 1.441 1.520 1.520 1.464 1.365 1.439 1.541 1.386 1.500 1.530 1.481 1.491 1.465 1.485 1.510 1.454 1.443 1.481 — — — 1.429 1.463 1.447 1.415 1.513 1.454 1.502 1.492 1.578 1.484 1.436 1.505 1.512 1.395 1.481 1.584 1.430 1.465 1.516 1.526 1.523 1.638 1.619 1.645 1.544 1.540

0.004 1.269 0.102 1.109 0.016 0.016 1.885 0.150 1.339 0.036 0.010 1.664 0.094 1.619 0.018 0.007 2.372 0.312 1.825 0.049 0.007 3.489 0.149 2.398 0.029 0.012 3.203 0.038 2.923 0.040 0.011 2.967 0.037 2.747 0.046 0.016 3.411 0.191 2.954 0.054 — — — — — — — — — — 0.017 3.791 0.140 3.170 0.045 0.014 3.431 0.073 3.314 0.091 0.010 3.591 0.079 3.358 0.034 0.014 4.087 0.170 3.148 0.040 0.016 3.554 0.094 3.008 0.343 0.014 3.352 0.269 2.789 0.062 0.007 4.139 0.197 3.077 0.029 0.019 3.639 0.053 3.300 0.067 0.026 3.854 0.049 2.976 0.257 0.008 5.110 0.109 4.243 0.030 0.017 4.958 0.133 4.521 0.103 0.021 4.602 0.079 4.529 0.082 0.019 6.086 0.106 5.549 0.138 0.015 4.350 0.094 4.757 0.117 0.012 3.764 0.126 3.354 0.034 0.066 10.474 0.690 8.818 0.281 0.041 9.615 0.458 9.452 0.244 0.039 7.752 0.174 7.599 0.282 0.070 16.013 0.928 13.906 0.659 — — — — — — — — — — — — — — — 0.047 9.200 0.262 10.366 0.341 0.026 8.382 0.464 8.576 0.325 0.034 8.163 0.269 8.177 0.185 0.037 6.873 0.205 6.566 0.134 0.020 5.571 0.093 5.115 0.059 0.017 6.116 0.098 5.519 0.104 0.040 5.774 0.113 5.553 0.187 0.026 4.948 0.056 4.163 0.064 0.022 4.955 0.206 4.264 0.050 0.013 3.647 0.064 3.642 0.082 0.026 4.640 0.213 3.577 0.074 0.023 4.490 0.144 3.839 0.034 0.023 4.230 0.103 3.429 0.045 0.013 4.018 0.078 3.563 0.087 0.016 3.883 0.060 3.187 0.039 0.015 4.082 0.101 3.410 0.053 0.017 3.080 0.113 2.985 0.041 0.020 3.356 0.074 3.156 0.076 0.024 3.709 0.084 3.651 0.102 0.018 4.993 0.157 4.072 0.019 0.019 5.621 0.133 4.243 0.032 0.030 4.831 0.141 4.812 0.082 0.018 5.522 0.091 4.938 0.116 0.027 6.116 0.182 5.705 0.109 0.018 5.787 0.125 5.211 0.081 0.009 5.921 0.154 4.902 0.040

PAGE S28

1

H-15N hetNOE

J(0)

J(0.87H)

J(N)

—

rad·ns-1

rad·ns-1

rad·ns-1

-1.593 -0.818 -0.778 -0.712 -0.244 -0.099 -0.053 -0.039 — — -0.122 -0.126 -0.129 -0.033 0.240 -0.200 0.026 -0.193 0.004 0.090 0.194 0.096 0.289 0.122 0.090 0.034 0.423 0.280 0.479 — — — 0.348 0.437 0.200 0.285 0.232 0.201 0.240 0.123 0.141 0.146 0.039 -0.011 0.047 0.006 -0.011 0.059 -0.051 0.080 0.120 0.010 0.139 0.310 0.237 0.083 0.183 0.108

0.209 0.353 0.273 0.464 0.767 0.677 0.617 0.733 — — 0.838 0.740 0.773 0.914 0.778 0.729 0.942 0.782 0.870 1.207 1.162 1.067 1.487 1.000 0.831 2.711 2.486 1.961 4.280 — — — 2.371 2.138 2.075 1.719 1.337 1.499 1.396 1.163 1.153 0.800 1.084 1.030 0.957 0.915 0.863 0.905 0.645 0.719 0.812 1.169 1.348 1.112 1.308 1.468 1.392 1.429

0.034 0.031 0.033 0.033 0.026 0.025 0.023 0.024 — — 0.026 0.025 0.027 0.024 0.017 0.026 0.022 0.029 0.022 0.021 0.019 0.021 0.017 0.020 0.021 0.023 0.013 0.016 0.012 — — — 0.015 0.013 0.018 0.016 0.018 0.018 0.018 0.020 0.021 0.020 0.022 0.024 0.022 0.022 0.023 0.023 0.023 0.021 0.021 0.024 0.020 0.018 0.019 0.024 0.020 0.021

0.107 0.156 0.172 0.183 0.215 0.232 0.228 0.238 — — 0.236 0.231 0.244 0.247 0.248 0.217 0.236 0.245 0.227 0.248 0.257 0.246 0.254 0.244 0.246 0.248 0.252 0.246 0.259 — — — 0.246 0.254 0.244 0.241 0.256 0.245 0.254 0.248 0.263 0.248 0.236 0.246 0.249 0.228 0.242 0.261 0.232 0.242 0.252 0.250 0.254 0.280 0.274 0.272 0.259 0.256

1

HN

15

N

H

1

H-15N RDC

R1

R1

R2

R2

R1

R1

[Hz]

[Hz]

[Hz]

[Hz]

1

[ppm]

[ppm]

[ppm]

[Hz]

[Hz]

[Hz]

N59 S60 R61 G62 W63 A64 N65 D66 G67 R68 T69 P70 G71 S72 R73 N74 L75 A76 N77 I78 P79 A80 S81 A82 L83 L84 S85 S86 D87 I88 T89 A90 S91 V92 N93 A94 A95 K96 K97 I98 V99 S100 D101 G102 N103 G104 M105 N106 A107 W108 V109 A110 W111 R112 N113 R114 C115 K116 G117

8.421 8.221 8.291 8.284 7.932 8.135 8.237 8.363 8.370 8.034 8.245 — 8.503 8.126 8.424 8.433 8.252 8.191 8.273 7.996 — 8.494 8.244 8.198 7.940 8.009 8.205 — 8.393 8.073 8.134 8.229 8.236 8.157 8.425 8.170 — 8.055 8.128 8.151 8.276 8.409 8.550 8.417 8.370 8.487 8.251 8.408 8.261 8.056 — — 7.953 — 8.041 7.810 8.092 — 8.338

122.330 117.013 122.873 109.826 121.545 125.534 118.109 119.282 109.202 120.609 117.799 — 110.059 116.156 123.265 120.217 123.658 124.046 117.872 123.265 — 125.141 114.125 126.314 120.765 122.330 116.388 — 121.477 121.860 118.264 126.469 115.840 122.640 122.386 124.908 — 120.062 122.795 123.343 125.218 120.062 122.015 109.904 119.370 109.904 120.764 119.752 124.438 120.377 — — 120.217 — 117.638 120.285 118.189 — 110.296

4.715 4.275 4.271 3.890 4.652 4.219 4.602 4.661 3.914 4.386 4.386 — 3.949 4.435 4.327 4.656 4.322 4.273 4.656 4.379 — 4.288 4.219 4.327 4.327 4.327 4.381 — 4.764 4.160 4.214 4.273 4.435 4.037 4.656 4.219 — 4.219 4.219 4.273 4.165 4.489 4.769 3.940 4.735 3.947 4.415 4.577 4.215 4.384 — — 4.445 — 4.494 4.214 4.607 — 4.002

-4.460 -3.488 -1.128 1.051 -0.688 -0.393 -2.448 -1.626 -1.247 -2.562 -2.962 — -4.006 -2.574 -5.188 -4.705 -4.160 -1.616 -3.986 -9.147 — 0.574 -2.077 0.787 -1.855 -2.004 -0.505 — -0.504 1.214 -1.080 0.431 0.279 0.255 -4.474 1.720 — -1.293 2.665 -5.621 -8.677 -6.566 -6.859 2.226 -1.583 0.290 3.073 4.066 3.144 -1.759 — — -1.513 — — — — — -2.807

1.570 1.499 1.612 1.571 1.593 1.596 1.514 1.530 1.561 1.567 1.466 — 1.416 1.477 1.478 1.518 1.466 1.495 1.434 1.409 — 1.439 1.472 1.567 1.475 1.495 1.464 — 1.505 1.490 1.505 1.461 1.342 1.426 1.570 1.417 — 1.412 1.447 1.421 1.431 1.440 1.473 1.386 1.530 1.420 1.450 1.443 1.407 1.398 — — 1.326 — 1.411 1.619 1.578 — 1.495

0.020 6.215 0.309 4.815 0.051 0.012 4.274 0.060 4.205 0.038 0.008 4.864 0.195 4.172 0.100 0.029 3.978 0.125 3.536 0.053 0.016 4.888 0.144 3.876 0.049 0.010 4.189 0.195 3.879 0.088 0.017 3.284 0.045 3.398 0.040 0.017 3.761 0.038 3.743 0.053 0.025 3.166 0.056 3.055 0.038 0.026 3.757 0.101 3.315 0.059 0.013 3.500 0.063 3.493 0.015 — — — — — 0.016 3.027 0.174 2.738 0.054 0.011 3.219 0.028 3.191 0.064 0.022 3.559 0.171 3.011 0.054 0.021 3.406 0.079 2.903 0.057 0.020 3.940 0.158 3.149 0.055 0.008 4.016 0.173 3.334 0.074 0.025 3.248 0.062 3.274 0.073 0.013 4.572 0.195 3.610 0.052 — — — — — 0.010 3.203 0.045 2.923 0.038 0.031 5.258 0.199 4.230 0.061 0.020 4.374 0.135 4.279 0.071 0.025 5.464 0.216 4.617 0.086 0.018 5.491 0.139 4.470 0.084 0.018 4.019 0.048 4.006 0.077 — — — — — 0.022 4.647 0.119 3.839 0.032 0.017 4.978 0.120 4.105 0.033 0.026 4.610 0.085 4.608 0.055 0.019 4.308 0.088 4.158 0.056 0.018 4.163 0.070 3.795 0.095 0.011 4.748 0.182 4.125 0.040 0.019 6.215 0.372 4.815 0.049 0.012 4.384 0.133 3.983 0.068 — — — — — 0.033 4.776 0.066 4.181 0.086 0.013 3.929 0.136 3.857 0.080 0.022 5.841 0.254 4.963 0.027 0.012 5.391 0.235 5.211 0.082 0.018 4.625 0.088 4.129 0.120 0.017 4.907 0.151 3.900 0.047 0.021 3.772 0.176 3.573 0.048 0.016 3.761 0.041 3.743 0.050 0.019 4.008 0.113 4.103 0.066 0.015 5.794 0.267 5.584 0.088 0.021 6.817 0.185 7.413 0.099 0.034 7.680 0.511 8.511 0.293 0.044 9.042 0.272 8.525 0.169 — — — — — — — — — — 0.053 9.747 0.649 11.062 0.857 — — — — — 0.040 11.567 0.302 11.920 0.693 0.022 10.368 0.611 7.710 0.654 0.066 6.689 0.392 8.203 0.431 — — — — — 0.034 8.489 0.362 7.364 0.179

T118

8.034

113.965

4.266

-1.223

1.519 0.022

8.718

0.316

7.429

PAGE S29

0.206

1

H-15N hetNOE

J(0)

J(0.87H)

J(N)

—

rad·ns-1

rad·ns-1

rad·ns-1

0.106 0.209 0.097 0.187 0.254 0.140 0.126 0.044 -0.029 0.003 0.094 — 0.027 0.015 -0.128 -0.107 0.018 0.060 0.043 0.043 — -0.099 0.213 0.163 0.194 0.058 0.059 — -0.011 -0.060 0.109 0.105 0.131 -0.039 0.106 0.019 — -0.064 -0.028 0.125 0.096 -0.089 0.085 0.029 0.013 0.082 0.387 0.326 0.538 0.452 — — 0.539 — 0.437 0.493 0.455 — 0.322

1.507 0.975 1.121 0.880 1.134 0.935 0.693 0.823 0.649 0.815 0.760 — 0.634 0.678 0.771 0.722 0.882 0.900 0.693 1.069 — 0.677 1.255 0.992 1.312 1.314 0.906 — 1.074 1.169 1.066 0.988 0.966 1.114 1.507 1.014 — 1.124 0.881 1.425 1.297 1.077 1.154 0.847 0.822 0.910 1.412 1.700 1.951 2.334 — — 2.544 — 3.041 2.674 1.646 — 2.162

0.022 0.018 0.023 0.020 0.019 0.021 0.021 0.023 0.025 0.024 0.021 — 0.021 0.023 0.026 0.026 0.022 0.022 0.021 0.021 — 0.025 0.018 0.020 0.019 0.022 0.022 — 0.024 0.025 0.021 0.020 0.018 0.023 0.022 0.022 — 0.023 0.023 0.019 0.020 0.024 0.021 0.021 0.024 0.020 0.014 0.015 0.010 0.012 — — 0.010 — 0.012 0.013 0.013 — 0.016

0.261 0.253 0.267 0.264 0.270 0.266 0.252 0.252 0.254 0.256 0.243 — 0.232 0.242 0.237 0.244 0.240 0.247 0.236 0.232 — 0.232 0.248 0.262 0.248 0.246 0.241 — 0.246 0.242 0.250 0.243 0.224 0.232 0.261 0.232 — 0.229 0.236 0.237 0.237 0.232 0.244 0.228 0.251 0.235 0.250 0.247 0.248 0.244 — — 0.234 — 0.245 0.284 0.275 — 0.256

0.301

2.222

0.017

0.259

1

HN

D119 V120 Q121 A122 W123 I124 R125 G126 A127 R128 L129

15

N

H

1

1

H-15N RDC

R1

R1

R2

R2

R1

R1

1

H-15N hetNOE

J(0)

J(0.87H)

J(N)

[ppm]

[ppm]

[ppm]

[Hz]

[Hz]

[Hz]

[Hz]

[Hz]

[Hz]

[Hz]

—

rad·ns

rad·ns

rad·ns-1

8.503 8.084 8.338 8.131 — 7.901 8.174 8.300 8.048 8.268 8.332

122.015 121.235 123.343 124.588 — 123.110 124.748 110.606 124.283 120.842 125.451

4.766 3.944 4.170 4.219 — 3.998 4.160 3.896 4.271 4.258 4.278

-2.978 -2.487 -3.350 -0.338 — -0.071 -0.789 -2.266 -0.315 -3.160 -1.502

1.527 1.591 1.553 1.415 — 1.621 1.545 1.443 1.433 1.262 1.128

0.031 0.042 0.057 0.056 — 0.029 0.026 0.029 0.008 0.008 0.015

8.418 8.460 7.911 9.452 — 8.703 7.547 4.525 3.971 3.492 2.313

0.322 0.203 0.289 0.460 — 0.248 0.247 0.146 0.183 0.105 0.091

6.378 9.009 7.210 7.375 — 7.843 6.849 4.318 3.382 2.967 2.219

0.121 0.126 0.155 0.277 — 0.232 0.168 0.056 0.070 0.087 0.042

0.264 0.209 0.217 0.174 — 0.323 0.196 -0.047 -0.145 -0.312 -0.628

2.135 2.137 1.988 2.441 — 2.203 1.887 1.049 0.893 0.782 0.468

0.018 0.020 0.019 0.018 — 0.017 0.019 0.024 0.026 0.026 0.029

0.259 0.268 0.262 0.237 — 0.278 0.260 0.234 0.229 0.197 0.168

PAGE S30

-1

-1

Table S4: NMR spectroscopic data of 1SS64-80-HEWL. R1. R2 and R1are fractional errors derived from fitting the exponential decay of intensity profiles in SPARKY. 1

HN

K1 V2 F3 G4 R5 S6 E7 L8 A9 A10 A11 M12 K13 R14 H15 G16 L17 D18 N19 Y20 R21 G22 Y23 S24 L25 G26 N27 W28 V29 A30 A31 A32 K33 F34 E35 S36 N37 F38 N39 T40 Q41 A42 T43 N44 R45 N46 T47 D48 G49 S50 T51 D52 Y53 G54 I55 L56 Q57 I58

15

N

H

1

H-15N RDC

R1

R1

R2

R2

R1

R1

1

1

H-15N hetNOE

J(0)

J(0.87H)

J(N)

[ppm]

[ppm]

[ppm]

[Hz]

[Hz]

[Hz]

[Hz]

[Hz]

[Hz]

[Hz]

—

rad·ns-1

rad·ns-1

rad·ns-1

8.713 8.261 8.535 8.376 8.314 8.509 8.416 8.189 8.229 8.110 8.157 8.134 8.128 8.205 8.518 8.463 8.235 8.541 8.330 8.088 8.252 7.782 7.969 8.283 8.229 8.243 8.212 8.102 7.751 8.062 8.101 8.133 8.103 8.134 8.173 8.291 8.369 8.174 — 8.134 8.345 8.299 8.118 8.395 8.353 8.517 8.197 8.518 8.400 8.197 8.220 8.393 8.142 8.283 7.868 8.236 8.298 8.134

125.657 123.320 125.948 111.487 121.542 117.708 123.506 123.649 124.900 122.747 123.012 119.365 122.664 122.071 120.057 110.853 122.356 120.164 119.857 121.412 124.224 109.542 120.608 118.398 124.799 109.274 119.506 122.561 123.138 127.348 123.466 123.486 120.408 121.290 122.459 117.436 120.905 121.105 — 115.222 122.826 125.617 113.519 121.680 122.154 120.349 114.769 121.621 110.215 116.245 116.334 121.775 121.577 110.398 120.408 125.542 122.095 122.257

4.380 4.101 4.648 3.935 4.327 4.327 4.377 4.327 4.270 4.217 4.219 4.399 4.344 4.270 4.715 3.968 4.377 4.653 4.653 4.502 4.218 3.826 4.587 4.419 4.318 3.837 4.711 4.601 3.842 4.110 4.110 4.218 4.176 4.594 4.323 4.323 4.597 4.652 — 4.272 4.298 4.323 4.321 4.707 4.331 4.762 4.326 4.707 3.993 4.487 4.272 4.707 4.487 3.886 4.164 4.331 4.323 4.055

-5.502 -7.375 -3.004 -0.710 -0.637 0.957 0.883 -0.462 -4.303 1.853 2.514 0.018 -0.824 -3.054 -4.426 -3.511 -2.932 -4.516 -3.535 0.067 -1.342 -2.904 -0.377 -2.719 -4.300 -1.907 -3.198 -2.788 2.208 0.487 -3.902 -0.457 -2.836 -2.297 -2.736 -2.406 -2.790 -0.433 — -3.172 -3.160 -0.856 -3.374 -4.279 -3.493 -2.562 -4.204 -2.376 -0.502 -1.558 -3.298 -3.739 -0.514 0.440 1.731 -1.850 -2.876 -0.994

0.878 1.143 1.243 1.278 1.419 1.507 1.488 1.564 1.562 1.574 1.556 1.579 1.539 1.583 1.565 1.438 1.517 1.475 1.522 1.588 1.652 1.556 1.592 1.550 1.615 1.476 1.503 1.565 1.572 1.523 1.466 1.488 1.474 1.510 1.522 1.472 1.507 1.501 — 1.471 1.536 1.463 1.409 1.482 1.489 1.552 1.445 1.540 1.438 1.440 1.476 1.587 1.581 1.648 1.605 1.686 1.590 1.612

0.004 0.002 0.005 0.005 0.010 0.007 0.010 0.005 0.008 0.012 0.009 0.010 0.005 0.010 0.011 0.012 0.010 0.013 0.020 0.011 0.006 0.008 0.010 0.015 0.010 0.008 0.003 0.008 0.006 0.011 0.016 0.012 0.012 0.006 0.006 0.009 0.014 0.011 — 0.015 0.008 0.007 0.015 0.025 0.008 0.009 0.018 0.015 0.013 0.015 0.017 0.016 0.019 0.019 0.012 0.040 0.011 0.025

0.935 1.279 1.319 1.417 1.573 2.033 2.244 2.361 2.408 2.465 2.684 2.545 2.831 2.593 2.614 2.114 2.490 2.468 2.429 2.632 2.723 3.058 2.799 2.640 2.707 — 2.835 3.176 3.656 — 3.124 3.094 3.257 3.053 3.500 3.561 2.766 2.696 — 2.514 2.891 2.500 2.349 2.607 2.655 2.583 2.437 2.879 2.348 2.462 2.911 3.762 4.523 4.478 4.742 — 6.017 7.758

0.038 0.060 0.044 0.079 0.052 0.069 0.087 0.083 0.059 0.079 0.077 0.083 0.083 0.069 0.075 0.058 0.084 0.085 0.052 0.083 0.114 0.217 0.097 0.080 0.111 — 0.094 0.100 0.133 — 0.136 0.131 0.107 0.082 0.119 0.098 0.062 0.079 — 0.070 0.058 0.107 0.079 0.085 0.087 0.067 0.074 0.074 0.073 0.061 0.088 0.139 0.102 0.149 0.149 — 0.188 0.275

1.036 1.195 1.394 1.589 1.752 2.105 2.245 2.330 2.541 2.587 2.636 2.483 2.647 2.648 2.702 2.219 2.436 2.450 2.466 2.811 2.873 2.622 2.883 2.680 2.794 2.625 2.828 3.074 3.174 3.499 3.238 3.117 3.258 3.221 3.370 3.483 2.759 2.785 — 2.585 2.808 2.488 2.468 2.668 2.606 2.637 2.504 2.659 2.377 2.554 2.952 3.233 4.158 4.441 4.677 — 5.757 7.008

0.028 0.012 0.024 0.027 0.040 0.025 0.038 0.024 0.051 0.023 0.052 0.052 0.055 0.030 0.088 0.028 0.030 0.110 0.046 0.044 0.072 0.033 0.064 0.020 0.072 0.048 0.075 0.016 0.174 0.044 0.048 0.041 0.051 0.021 0.039 0.041 0.015 0.015 — 0.024 0.027 0.023 0.134 0.045 0.031 0.079 0.029 0.037 0.051 0.041 0.032 0.058 0.061 0.116 0.098 — 0.088 0.115

-1.397 -0.717 -0.616 -0.610 -0.092 -0.144 0.030 0.044 0.084 -0.080 -0.061 -0.092 -0.080 0.031 -0.091 -0.159 0.039 0.002 -0.011 -0.072 0.128 -0.090 0.173 0.078 0.084 0.050 0.075 -0.109 -0.072 -0.057 -0.097 -0.069 -0.114 -0.074 0.035 0.083 -0.071 -0.022 — -0.129 -0.104 -0.017 -0.157 -0.088 -0.101 0.027 -0.029 -0.026 -0.148 0.067 0.129 -0.019 -0.038 0.267 0.224 0.217 0.248 0.243

0.112 0.173 0.170 0.192 0.222 0.337 0.403 0.424 0.439 0.449 0.514 0.471 0.558 0.486 0.492 0.371 0.468 0.467 0.449 0.494 0.514 0.619 0.546 0.506 0.515 — 0.567 0.650 0.785 — 0.651 0.640 0.687 0.624 0.750 0.776 0.545 0.527 — 0.478 0.575 0.478 0.441 0.503 0.516 0.488 0.462 0.572 0.436 0.472 0.594 0.813 1.027 1.011 1.090 — 1.451 1.937

0.033 0.031 0.031 0.032 0.024 0.027 0.023 0.023 0.022 0.027 0.026 0.027 0.026 0.024 0.027 0.026 0.023 0.023 0.024 0.027 0.023 0.026 0.021 0.022 0.023 — 0.022 0.027 0.026 — 0.025 0.025 0.026 0.025 0.023 0.021 0.025 0.024 — 0.026 0.026 0.023 0.025 0.025 0.026 0.024 0.023 0.025 0.026 0.021 0.020 0.025 0.026 0.019 0.019 — 0.019 0.019

0.115 0.167 0.185 0.191 0.229 0.241 0.244 0.257 0.259 0.254 0.252 0.255 0.249 0.260 0.253 0.230 0.249 0.241 0.249 0.257 0.275 0.251 0.267 0.256 0.267 — 0.248 0.252 0.254 — 0.236 0.241 0.237 0.244 0.250 0.244 0.244 0.245 — 0.236 0.247 0.239 0.225 0.239 0.240 0.255 0.235 0.251 0.230 0.238 0.246 0.259 0.257 0.280 0.271 — 0.269 0.273

PAGE S31

1

HN

15

N

H

1

H-15N RDC

R1

R1

R2

R2

R1

R1

[Hz]

[Hz]

[Hz]

[Hz]

[Hz]

—

—

—

1

[ppm]

[ppm]

[ppm]

[Hz]

N59

8.423

122.399

4.707

-2.839

1.407 0.007

1

H-15N hetNOE

J(0)

J(0.87H)

J(N)

[Hz]

—

rad·ns-1

rad·ns-1

rad·ns-1

—

-0.194

—

—

—

S60

8.209

116.589

4.268

-2.519

1.579 0.043

—

—

—

—

0.251

—

—

—

R61 W62 W63 C64 N65 D66 G67 R68 T69 P70 G71 S72 R73 N74 L75 A76 N77 I78 P79 C80 S81 A82 L83 L84 S85 S86 D87 I88 T89 A90 S91 V92 N93 A94 A95 K96 K97 I98 V99 S100 D101 G102 N103 G104 M105 N106 A107 W108 V109 A110 W111 R112 N113 R114 A115 K116 G117

8.157 7.945 7.907 — — 8.393 8.385 8.025 8.236 — 8.533 8.032 8.471 8.517 — 8.102 8.503 — — — 8.447 8.220 7.978 8.032 8.189 8.205 8.394 8.078 8.165 8.252 8.283 8.182 8.431 8.213 8.157 8.125 8.229 8.244 8.330 8.448 8.588 8.424 8.370 8.486 8.228 8.416 8.213 8.056 7.767 8.040 7.909 7.976 8.112 7.939 8.196 8.237 8.385

123.852 122.115 121.305 — — 120.656 109.086 120.432 117.781 — 110.236 116.055 123.178 120.530 — 122.953 120.203 — — — 117.332 126.361 121.046 122.636 116.616 117.065 121.538 122.008 118.664 126.861 116.104 122.707 122.237 125.353 123.241 120.842 123.443 124.021 125.887 120.427 122.214 110.057 119.306 109.992 120.573 120.023 124.528 120.452 121.866 125.427 119.857 121.763 118.790 121.743 125.184 121.188 110.503

4.323 4.648 4.599 — — 4.648 3.894 4.430 4.421 — 3.943 4.433 4.407 4.638 — 4.293 4.711 — — — 4.329 4.329 4.272 4.279 4.331 4.382 4.724 4.192 4.217 4.272 4.331 4.085 4.653 4.269 4.268 4.255 4.273 4.154 4.165 4.486 4.760 3.952 4.707 3.952 4.433 4.600 4.219 4.546 3.778 4.105 4.486 3.945 4.600 4.219 4.217 4.272 4.001

-1.664 -1.436 0.839 — — 1.460 -0.170 2.064 -0.622 — -3.046 -0.386 -2.301 -2.862 — -0.796 -1.459 — — — -0.366 -0.283 -1.043 -1.809 -1.161 -3.101 -1.373 -0.938 -3.523 -1.427 -1.487 -2.128 -2.358 -1.558 0.739 -3.021 -6.377 -5.229 -4.971 -4.374 -4.842 0.304 -2.076 -1.884 0.079 -0.322 0.323 6.215 7.157 3.366 0.544 4.588 2.696 -7.808 -1.157 -2.951 -5.098

1.564 1.646 1.661 — — 1.658 1.750 1.688 1.476 — 1.551 1.601 1.646 1.736 — 1.632 1.630 — — — 1.725 1.640 1.599 1.485 1.620 1.583 1.527 1.487 1.479 1.433 1.314 1.389 1.407 1.397 1.388 1.377 1.438 1.443 1.408 1.370 1.372 1.341 1.406 1.386 1.511 1.508 1.624 1.679 1.733 1.784 1.750 1.794 1.763 1.675 1.596 1.527 1.478

0.006 0.026 0.063 — — 0.029 0.013 0.017 0.015 — 0.025 0.019 0.021 0.028 — 0.022 0.050 — — — 0.026 0.041 0.013 0.015 0.024 0.018 0.013 0.009 0.014 0.010 0.006 0.006 0.007 0.010 0.009 0.010 0.008 0.004 0.016 0.009 0.012 0.006 0.004 0.009 0.006 0.008 0.005 0.012 0.015 0.017 0.007 0.016 0.013 0.009 0.008 0.003 0.012

9.833 6.169 8.688 — — — 6.739 5.081 4.036 — 3.962 4.155 4.237 — — 4.980 6.394 — — — 8.658 6.954 5.893 5.801 4.401 — 3.333 3.757 3.934 3.978 3.077 3.230 3.399 3.056 2.738 3.139 3.345 3.224 3.234 2.801 2.462 2.065 2.254 2.229 2.769 2.901 3.255 3.909 4.268 4.575 4.270 4.223 3.953 3.322 2.802 2.706 2.266

0.586 0.200 0.278 — — — 0.201 0.182 0.119 — 0.147 0.135 0.205 — — 0.394 0.363 — — — 0.389 0.127 0.198 0.182 0.169 — 0.117 0.123 0.112 0.269 0.099 0.122 0.094 0.113 0.130 0.089 0.158 0.130 0.095 0.082 0.080 0.113 0.068 0.066 0.072 0.101 0.091 0.098 0.146 0.167 0.107 0.103 0.147 0.100 0.130 0.092 0.092

8.347 5.669 8.217 — — — 5.136 5.003 3.890 — 3.945 4.376 4.243 — — 4.562 6.817 — — — 8.320 6.519 5.838 5.423 4.365 — 3.667 3.498 3.867 3.427 3.057 3.129 3.349 3.100 2.954 3.266 3.151 3.154 3.254 2.823 2.337 2.201 2.290 2.254 2.826 3.069 3.432 3.920 4.283 4.684 4.259 4.072 3.965 3.434 2.799 2.906 2.329

0.226 0.119 0.600 — — — 0.063 0.060 0.069 — 0.079 0.067 0.141 — — 0.071 0.333 — — — 0.241 0.138 0.124 0.138 0.083 — 0.059 0.034 0.074 0.049 0.032 0.029 0.024 0.047 0.064 0.034 0.029 0.184 0.071 0.028 0.046 0.075 0.038 0.026 0.054 0.027 0.028 0.036 0.062 0.061 0.063 0.049 0.052 0.028 0.030 0.036 0.042

0.208 0.481 0.286 — — 0.430 0.363 0.183 0.183 — 0.310 0.309 0.217 0.383 — 0.220 0.163 — — — 0.501 0.446 0.334 0.237 0.326 0.134 -0.036 -0.067 -0.013 0.059 -0.049 -0.088 -0.194 0.005 -0.125 -0.228 -0.103 -0.043 -0.162 -0.320 -0.021 -0.292 -0.172 -0.175 0.073 -0.107 0.166 0.187 0.180 0.180 0.335 0.289 0.150 0.238 0.055 0.124 -0.174

2.526 1.491 2.192 — — — 1.633 1.172 0.911 — 0.881 0.928 0.943 — — 1.153 1.550 — — — 2.179 1.711 1.417 1.406 0.995 — 0.701 0.826 0.878 0.899 0.662 0.693 0.736 0.644 0.554 0.667 0.717 0.683 0.690 0.571 0.481 0.369 0.414 0.411 0.547 0.582 0.669 0.845 0.937 1.016 0.939 0.918 0.843 0.682 0.543 0.529 0.406

0.019 0.013 0.019 — — — 0.017 0.022 0.019 — 0.017 0.017 0.020 — — 0.020 0.021 — — — 0.013 0.014 0.017 0.018 0.017 — 0.025 0.025 0.023 0.021 0.022 0.024 0.026 0.022 0.024 0.026 0.025 0.024 0.026 0.028 0.022 0.027 0.026 0.025 0.022 0.026 0.021 0.021 0.022 0.023 0.018 0.020 0.023 0.020 0.024 0.021 0.027

0.264 0.288 0.283 — — — 0.301 0.283 0.248 — 0.265 0.274 0.278 — — 0.275 0.273 — — — 0.303 0.286 0.274 0.251 0.277 — 0.248 0.241 0.241 0.236 0.213 0.224 0.224 0.229 0.223 0.218 0.232 0.235 0.225 0.214 0.224 0.210 0.224 0.221 0.250 0.243 0.272 0.282 0.291 0.299 0.300 0.306 0.295 0.283 0.263 0.254 0.236

T118

8.079

113.930

4.288

-2.529

1.468 0.007

2.565

0.088

2.472

0.013

-0.076

0.494

0.025

0.238

PAGE S32

1

HN

15

N

H

1

H-15N RDC

R1

R1

R2

R2

R1

R1

[Hz]

[Hz]

[Hz]

[Hz]

[Hz]

1

1

H-15N hetNOE

J(0)

J(0.87H)

J(N)

[Hz]

—

rad·ns-1

rad·ns-1

rad·ns-1

[ppm]

[ppm]

[ppm]

[Hz]

D119 V120

8.572 8.110

122.316 121.601

4.760 3.998

-4.198 -2.097

1.489 0.014 1.506 0.005

2.347 2.672

0.089 0.062

2.428 2.554

0.040 0.043

0.027 -0.120

0.431 0.517

0.023 0.026

0.244 0.242

Q121

8.377

124.061

4.219

-3.748

1.590 0.010

2.655

0.085

2.773

0.030

-0.185

0.498

0.029

0.253

A122

—

—

—

—

—

—

—

—

—

—

—

—

—

—

W123 I124 R125 G126 A127 R128 L129

8.001 7.885 — 8.337 8.078 8.299 8.368

120.510 123.669 — 110.770 124.406 121.085 125.678

4.653 3.998 — 3.944 4.272 4.272 4.272

1.601 1.984 — -5.242 -0.438 -4.236 -1.779

1.590 1.596 — 1.412 1.340 1.187 1.020

0.015 0.010 — 0.014 0.007 0.004 0.005

2.659 2.720 — 2.155 1.751 1.433 1.283

0.095 0.092 — 0.075 0.056 0.076 0.038

2.685 2.847 — 2.136 1.871 1.432 1.367

0.041 0.046 — 0.049 0.014 0.054 0.020

0.037 -0.002 — -0.278 -0.522 -0.664 -1.131

0.504 0.519 — 0.384 0.277 0.210 0.189

0.024 0.025 — 0.028 0.032 0.031 0.034

0.261 0.261 — 0.222 0.203 0.176 0.139

PAGE S33

Table S5: NMR spectroscopic data of 1SS76-94-HEWL. R1. R2 and R1are fractional errors derived from fitting the exponential decay of intensity profiles in SPARKY. 1

HN

K1 V2 F3 G4 R5 S6 E7 L8 A9 A10 A11 M12 K13 R14 H15 G16 L17 D18 N19 Y20 R21 G22 Y23 S24 L25 G26 N27 W28 V29 A30 A31 A32 K33 F34 E35 S36 N37 F38 N39 T40 Q41 A42 T43 N44 R45 N46 T47 D48 G49 S50 T51 D52 Y53 G54 I55 L56 Q57 I58

15

N

H

1

H-15N RDC

R1

R1

R2

R2

R1

R1

1

1

H-15N hetNOE

J(0)

J(0.87H)

J(N)

[ppm]

[ppm]

[ppm]

[Hz]

[Hz]

[Hz]

[Hz]

[Hz]

[Hz]

[Hz]

—

rad·ns-1

rad·ns-1

rad·ns-1

8.712 8.250 8.531 8.375 8.311 8.508 8.415 8.189 8.229 8.103 8.148 8.124 8.115 8.194 8.508 8.453 8.226 8.532 8.320 8.086 8.250 7.781 7.961 8.273 8.227 8.242 8.203 8.093 7.741 8.054 8.093 8.125 8.094 8.124 8.171 8.282 8.367 8.172 8.367 8.132 8.345 8.296 8.117 8.391 8.344 8.516 8.196 8.508 8.399 8.195 8.219 8.383 8.148 8.297 7.851 8.217 8.273 8.093

125.630 123.284 125.942 111.448 121.528 117.659 123.482 123.623 124.824 122.718 122.933 119.302 122.582 121.995 119.965 110.822 122.345 120.160 119.807 121.370 124.182 109.533 120.590 118.362 124.807 109.258 119.497 122.544 123.089 127.309 123.440 123.440 120.394 121.253 122.429 117.425 120.901 121.097 120.796 115.195 122.815 125.592 113.477 121.677 122.152 120.355 114.766 121.645 110.196 116.254 116.330 121.764 121.604 110.431 120.237 125.550 121.957 122.152

4.381 4.089 4.652 3.940 4.331 4.388 4.395 4.332 — 4.214 4.216 4.228 4.343 4.268 4.699 3.951 4.357 4.635 4.635 4.496 4.204 3.824 4.572 4.444 4.317 3.824 4.673 4.583 3.837 4.090 4.116 4.204 4.192 4.585 4.331 4.331 4.634 4.634 4.710 4.279 4.266 4.330 4.317 4.710 4.305 4.773 4.330 4.723 3.988 4.482 4.266 4.710 4.469 3.886 4.292 4.292 4.317 4.051

-6.242 -8.339 -3.562 -0.742 -0.851 0.935 1.135 -0.594 -4.708 2.211 2.873 0.012 -1.045 -3.206 -5.038 -3.607 -3.478 -4.880 -3.896 -0.090 -1.126 -3.499 -0.390 -3.089 0.264 -1.882 -3.565 -3.088 2.295 0.246 -4.383 -0.731 -3.721 -3.003 -2.738 -2.294 -3.200 -0.808 -4.307 -3.798 -3.592 -1.809 -3.817 -5.140 -3.888 -2.870 -4.480 -2.685 -0.378 -1.715 -3.730 -4.239 0.453 0.640 -2.179 -2.710 -3.211 -1.629

0.835 1.149 1.167 1.192 1.323 1.342 1.480 1.522 — 1.500 1.468 1.519 1.503 1.540 1.519 1.404 1.478 1.465 1.516 1.550 1.600 1.530 1.543 1.505 1.500 1.465 1.491 1.503 1.565 1.548 1.448 1.411 1.467 1.495 1.502 1.428 — 1.487 — 1.457 1.509 1.436 1.384 1.468 1.462 1.553 1.413 1.506 1.344 1.414 1.464 1.555 1.571 1.611 1.669 1.641 1.599 1.593

0.006 0.012 0.011 0.009 0.013 0.010 0.025 0.026 — 0.014 0.011 0.011 0.011 0.012 0.016 0.010 0.014 0.012 0.010 0.012 0.008 0.010 0.010 0.006 0.014 0.012 0.009 0.014 0.010 0.020 0.022 0.010 0.017 0.024 0.011 0.012 — 0.009 — 0.018 0.010 0.015 0.014 0.017 0.017 0.018 0.008 0.012 0.025 0.010 0.026 0.009 0.015 0.016 0.015 0.024 0.014 0.015

0.964 1.637 1.388 1.452 2.011 2.142 2.234 2.292 — 2.441 2.810 2.573 2.550 2.717 2.685 2.205 2.786 2.392 2.592 2.670 2.870 2.619 2.786 2.817 2.650 2.425 2.794 3.431 3.563 — 2.960 3.176 3.445 3.156 3.032 2.893 — 2.866 — 2.551 2.436 2.428 2.118 2.668 2.448 2.737 2.400 2.648 2.165 2.651 2.889 3.196 4.006 3.654 4.621 4.415 4.323 4.558

0.027 0.198 0.124 0.037 0.042 0.073 0.113 0.128 — 0.061 0.139 0.043 0.087 0.064 0.067 0.111 0.148 0.053 0.054 0.070 0.043 0.196 0.142 0.077 0.063 0.104 0.029 0.139 0.085 — 0.071 0.055 0.138 0.040 0.093 0.045 — 0.064 — 0.072 0.072 0.081 0.108 0.081 0.127 0.046 0.062 0.049 0.089 0.075 0.053 0.051 0.079 0.111 0.087 0.087 0.065 0.156

0.955 1.617 1.357 1.596 1.865 2.071 2.235 2.674 — 2.447 2.677 2.651 2.815 2.704 2.599 2.287 2.396 2.499 2.385 2.666 2.980 2.866 2.904 2.779 2.701 2.754 2.788 2.815 3.561 3.364 3.077 3.171 3.244 3.156 3.222 2.750 — 2.861 — 2.556 2.678 2.489 2.421 2.450 2.571 2.687 2.456 2.608 2.426 2.466 2.714 3.382 3.834 3.888 4.686 4.517 4.525 4.666

0.020 0.028 0.039 0.041 0.052 0.019 0.117 0.063 — 0.070 0.053 0.035 0.164 0.047 0.193 0.046 0.044 0.060 0.079 0.033 0.058 0.150 0.051 0.033 0.044 0.055 0.021 0.172 0.109 0.045 0.088 0.092 0.082 0.028 0.045 0.109 — 0.057 — 0.054 0.048 0.040 0.060 0.064 0.036 0.095 0.059 0.031 0.095 0.028 0.050 0.083 0.111 0.301 0.134 0.138 0.102 0.071

-1.497 -0.507 -0.687 -0.893 -0.290 -0.345 -0.429 -0.177 — -0.049 -0.151 -0.186 -0.136 -0.007 -0.088 -0.210 0.024 -0.087 -0.040 -0.180 0.118 -0.156 0.148 0.077 0.078 0.006 -0.048 -0.136 -0.164 -0.138 -0.123 -0.074 -0.156 -0.111 -0.025 -0.037 — -0.031 — -0.161 -0.193 0.137 -0.303 -0.217 -0.085 -0.004 -0.071 -0.089 -0.237 0.001 0.087 -0.217 -0.009 0.178 0.214 0.257 0.158 0.120

0.127 0.276 0.201 0.211 0.357 0.390 0.392 0.407 — 0.455 0.561 0.486 0.483 0.527 0.520 0.400 0.556 0.446 0.495 0.509 0.563 0.498 0.549 0.562 0.516 0.457 0.555 0.730 0.757 — 0.607 0.674 0.740 0.655 0.621 0.593 — 0.577 — 0.490 0.449 0.464 0.377 0.520 0.462 0.530 0.456 0.511 0.398 0.528 0.589 0.655 0.884 0.783 1.047 0.994 0.973 1.038

0.033 0.027 0.031 0.035 0.027 0.028 0.033 0.028 — 0.025 0.026 0.028 0.027 0.024 0.026 0.027 0.023 0.025 0.025 0.029 0.022 0.028 0.021 0.022 0.022 0.023 0.024 0.027 0.028 — 0.025 0.024 0.027 0.026 0.024 0.023 — 0.024 — 0.026 0.028 0.019 0.028 0.028 0.025 0.024 0.024 0.026 0.026 0.022 0.021 0.030 0.025 0.021 0.021 0.019 0.021 0.022

0.107 0.174 0.172 0.170 0.207 0.209 0.227 0.243 — 0.244 0.235 0.242 0.241 0.252 0.245 0.223 0.243 0.237 0.247 0.247 0.266 0.245 0.258 0.249 0.248 0.240 0.242 0.241 0.250 — 0.233 0.228 0.234 0.241 0.245 0.232 — 0.242 — 0.233 0.240 0.240 0.216 0.233 0.236 0.254 0.229 0.243 0.212 0.231 0.242 0.246 0.257 0.270 0.281 0.278 0.267 0.265

PAGE S34

1

HN

15

N

H

1

H-15N RDC

R1

R1

R2

R2

R1

R1

1

1

H-15N hetNOE

J(0)

J(0.87H)

J(N)

[ppm]

[ppm]

[ppm]

[Hz]

[Hz]

[Hz]

[Hz]

[Hz]

[Hz]

[Hz]

—

rad·ns-1

rad·ns-1

rad·ns-1

N59 S60 R61 W62 W63 A64 N65 D66 G67 R68 T69 P70 G71 S72 R73 N74 L75 C76 N77 I78 P79 A80 S81 A82 L83 L84 S85 S86 D87 I88 T89 A90 S91 V92 N93 C94 A95 K96 K97 I98 V99 S100 D101 G102 N103 G104 M105 N106 A107 W108 V109 A110 W111 R112 N113 R114 A115 K116 G117

8.352 8.211 8.227 7.820 7.389 7.718 8.117 8.328 8.335 7.851 8.211 — 8.335 8.180 8.329 8.430 — — — 7.866 — 8.524 8.188 8.070 7.968 8.391 8.141 8.131 8.336 8.038 8.102 8.118 8.117 8.070 8.383 8.274 8.227 8.149 — 8.233 8.297 8.422 8.563 8.414 8.366 8.477 8.226 8.407 8.212 8.055 7.788 8.031 7.912 7.984 8.110 7.937 8.179 8.227 8.376

122.269 116.996 122.853 121.331 121.292 124.731 117.739 119.259 109.065 120.394 117.465 — 109.215 115.163 123.482 120.356 — — — 122.777 — 125.123 112.854 125.904 121.487 123.170 116.135 115.743 120.862 121.487 117.385 125.942 117.148 121.919 117.814 119.807 125.356 120.706 — 123.752 125.474 120.199 122.152 110.003 119.302 109.924 120.551 119.964 124.461 120.433 121.761 125.318 119.883 121.723 118.792 121.723 125.047 121.136 110.471

4.710 4.254 4.254 4.495 4.482 4.152 4.621 4.634 3.874 4.165 4.381 — 3.886 4.343 4.330 4.710 — — — 4.419 — 4.152 4.203 4.305 4.292 4.381 4.419 4.368 4.761 4.165 4.216 4.279 4.419 4.102 4.710 4.697 4.279 4.266 — 4.266 4.178 4.482 4.773 3.937 4.723 3.950 4.419 4.583 4.203 4.545 3.772 4.114 4.495 3.937 4.583 4.216 4.216 4.228 4.000

-3.139 -1.068 -2.334 7.696 8.614 7.945 -3.559 -2.039 0.972 0.390 -2.747 — -1.170 -1.934 -5.193 -4.251 — — — -5.674 — -2.354 -2.706 1.426 -3.010 -4.677 -2.237 0.097 -2.099 -0.647 -2.297 -0.348 0.238 -3.144 -5.993 -3.814 -3.189 -4.276 — -6.131 -1.608 -4.381 -4.781 0.114 -1.867 -2.043 -0.106 0.116 0.802 5.012 6.282 3.148 1.170 3.820 2.708 -6.957 -1.410 -3.031 -4.913

1.568 1.544 1.686 1.692 1.706 1.696 1.581 1.579 1.584 1.603 1.507 — — 1.601 — 1.573 — — — 1.688 — 1.685 1.596 1.724 1.651 1.588 1.486 1.574 1.484 1.681 1.685 1.702 1.587 1.667 1.750 1.734 1.703 1.578 — 1.563 1.447 1.480 1.401 1.349 1.439 1.397 1.504 1.472 1.630 1.613 1.732 1.748 1.740 1.757 1.695 1.623 1.574 1.518 1.425

0.020 0.016 0.010 0.024 0.037 0.033 0.027 0.020 0.014 0.017 0.020 — — 0.021 — 0.030 — — — 0.019 — 0.017 0.014 0.023 0.025 0.009 0.017 0.012 0.008 0.023 0.032 0.010 0.032 0.024 0.040 0.019 0.020 0.015 — 0.023 0.017 0.026 0.019 0.014 0.006 0.014 0.009 0.022 0.023 0.010 0.028 0.024 0.021 0.024 0.010 0.017 0.011 0.014 0.011

4.617 5.516 4.724 5.534 6.234 4.980 4.462 4.314 4.702 4.078 4.044 — — 4.737 5.498 4.880 — — — 5.441 — 5.176 4.653 5.217 4.780 3.785 3.754 4.521 3.296 4.575 5.291 4.805 4.531 5.498 7.262 6.204 5.659 5.692 — 4.764 4.983 3.573 3.206 2.522 2.907 2.918 3.376 3.672 3.797 4.594 4.785 5.272 5.045 4.958 4.564 3.857 3.120 3.346 2.666

0.284 0.094 0.144 0.244 0.256 0.148 0.071 0.048 0.420 0.147 0.050 — — 0.104 0.232 0.097 — — — 0.134 — 0.080 0.089 0.198 0.254 0.118 0.101 0.040 0.047 0.066 0.092 0.138 0.060 0.101 0.101 0.208 0.333 0.095 — 0.114 0.281 0.086 0.076 0.097 0.047 0.061 0.036 0.118 0.100 0.129 0.138 0.118 0.079 0.102 0.037 0.199 0.145 0.111 0.059

5.035 5.255 4.838 5.385 5.640 5.308 4.221 4.075 3.817 4.042 3.775 — — 4.688 — 4.632 — — — 5.394 — 5.043 5.147 4.789 4.833 4.132 3.949 4.583 3.098 4.478 4.958 4.556 4.365 5.283 6.920 5.624 5.319 5.456 — 4.713 4.501 3.731 3.209 2.612 2.821 2.871 3.288 3.600 3.866 4.367 5.128 5.071 4.726 5.025 4.456 4.050 3.050 3.097 2.401

0.099 0.072 0.087 0.065 0.178 0.130 0.035 0.082 0.119 0.101 0.103 — — 0.041 — 0.118 — — — 0.113 — 0.066 0.116 0.040 0.129 0.078 0.111 0.059 0.027 0.094 0.288 0.065 0.050 0.075 0.197 0.158 0.099 0.082 — 0.054 0.103 0.121 0.082 0.081 0.029 0.033 0.030 0.153 0.081 0.075 0.119 0.115 0.150 0.137 0.059 0.069 0.033 0.070 0.108

-0.085 0.318 0.315 0.333 0.291 0.247 0.190 -0.117 0.162 0.214 0.188 — — 0.251 — 0.113 — — — 0.413 — 0.351 0.402 0.263 0.266 0.184 0.136 0.153 -0.049 0.173 0.227 0.181 0.122 0.263 0.530 0.426 0.421 0.307 — 0.173 -0.074 -0.094 -0.002 -0.214 -0.117 -0.005 0.123 -0.085 0.270 0.136 0.191 0.218 0.359 0.310 0.161 0.255 0.047 0.101 -0.167

1.055 1.319 1.075 1.302 1.496 1.144 1.015 0.967 1.081 0.904 0.908 — — 1.090 — 1.132 — — — 1.278 — 1.203 1.071 1.207 1.095 0.824 0.829 1.032 0.697 1.031 1.233 1.093 1.032 1.294 1.784 1.486 1.338 1.363 — 1.102 1.176 0.775 0.686 0.498 0.594 0.605 0.720 0.804 0.822 1.046 1.083 1.218 1.159 1.130 1.026 0.840 0.636 0.709 0.527

0.027 0.016 0.018 0.018 0.019 0.020 0.020 0.028 0.021 0.020 0.019 — — 0.019 — 0.022 — — — 0.016 — 0.017 0.015 0.020 0.019 0.020 0.020 0.021 0.024 0.022 0.020 0.022 0.022 0.019 0.013 0.016 0.015 0.017 — 0.020 0.024 0.025 0.022 0.026 0.025 0.022 0.021 0.025 0.019 0.022 0.022 0.021 0.017 0.019 0.022 0.019 0.023 0.021 0.026

0.253 0.264 0.288 0.290 0.291 0.287 0.266 0.254 0.265 0.270 0.253 — — 0.271 — 0.262 — — — 0.293 — 0.290 0.276 0.293 0.280 0.267 0.248 0.263 0.241 0.282 0.285 0.286 0.264 0.283 0.308 0.301 0.296 0.270 — 0.262 0.234 0.239 0.229 0.214 0.231 0.228 0.250 0.238 0.277 0.269 0.291 0.295 0.299 0.300 0.284 0.275 0.259 0.252 0.227

T118

8.078

113.907

4.279

-2.796

1.443 0.012

2.675

0.021

2.516

0.063

-0.148

0.527

0.026

0.231

PAGE S35

1

HN

D119 V120 Q121 A122 W123 I124 R125 G126 A127 R128 L129

15

N

H

1

1

H-15N RDC

R1

R1

R2

R2

R1

R1

1

H-15N hetNOE

J(0)

J(0.87H)

J(N)

[ppm]

[ppm]

[ppm]

[Hz]

[Hz]

[Hz]

[Hz]

[Hz]

[Hz]

[Hz]

—

rad·ns

rad·ns

rad·ns-1

8.563 8.109 8.368 — 8.000 7.882 8.187 8.329 8.070 8.296 8.345

122.307 121.604 123.950 — 120.472 123.558 125.161 110.706 124.381 121.058 125.786

4.761 3.975 4.190 — 4.634 4.000 4.216 3.937 4.279 4.279 4.292

-4.350 -2.152 -3.695 — 1.033 1.450 -1.410 -5.259 -0.444 -4.233 -1.751

1.540 1.477 1.569 — 1.559 1.582 1.658 1.381 1.341 1.150 1.021

0.016 0.012 0.011 — 0.012 0.017 0.014 0.007 0.023 0.008 0.008

2.174 2.943 2.963 — 3.204 2.975 3.197 2.373 1.825 1.788 1.385

0.223 0.164 0.058 — 0.050 0.130 0.197 0.029 0.071 0.041 0.030

2.650 2.684 3.115 — 3.170 3.075 3.499 2.387 1.867 1.770 1.417

0.080 0.032 0.040 — 0.040 0.075 0.095 0.017 0.043 0.038 0.034

0.083 -0.131 -0.172 — 0.059 0.018 0.047 -0.327 -0.590 -0.656 -1.163

0.376 0.598 0.588 — 0.662 0.594 0.645 0.449 0.297 0.316 0.217

0.022 0.026 0.029 — 0.023 0.024 0.025 0.029 0.033 0.030 0.035

0.255 0.237 0.250 — 0.257 0.259 0.273 0.215 0.201 0.170 0.139

PAGE S36

-1

-1

Video S1: Movie of a 360° rotation of a model of 1SS6-127-HEWL as used in Figure 5. The same color coding has been used. The movie images were generated with PyMOL (www.pymol.org, PDB ID: 6LYZ) and subsequently converted with the ffmpeg software package (www.ffmpeg.org)

Video S2: Movie of a 360° rotation of a model of 1SS30-115-HEWL as used in Figure 5. The same color coding has been used. The movie images were generated with PyMOL (www.pymol.org, PDB ID: 6LYZ) and subsequently converted with the ffmpeg software package (www.ffmpeg.org)

Video S3: Movie of a 360° rotation of a model of 1SS30-115W62G-HEWL as used in Figure 5. The same color coding has been used. The movie images were generated with PyMOL (www.pymol.org, PDB ID: 6LYZ) and subsequently converted with the ffmpeg software package (www.ffmpeg.org)

Video S4: Movie of a 360° rotation of a model of 1SS64-80-HEWL as used in Figure 5. The same color coding has been used. The movie images were generated with PyMOL (www.pymol.org, PDB ID: 6LYZ) and subsequently converted with the ffmpeg software package (www.ffmpeg.org)

Video S5: Movie of a 360° rotation of a model of 1SS76-94-HEWL as used in Figure 5. The same color coding has been used. The movie images were generated with PyMOL (www.pymol.org, PDB ID: 6LYZ) and subsequently converted with the ffmpeg software package (www.ffmpeg.org)

Video S6: Movie of a 360° rotation of a model of 2SS-HEWL as used in Figure 5. The same color coding has been used. The movie images were generated with PyMOL (www.pymol.org, PDB ID: 6LYZ) and subsequently converted with the ffmpeg software package (www.ffmpeg.org)

Video S7: Movie of a 360° rotation of a model of 2SS-HEWL as used in Figure 5. The same color coding has been used. The movie images were generated with PyMOL (www.pymol.org, PDB ID: 6LYZ) and subsequently converted with the ffmpeg software package (www.ffmpeg.org)

PAGE S37

References (1) Schlorb, C.; Ackermann, K.; Richter, C.; Wirmer, J.; Schwalbe, H. J Biomol NMR 2005, 33, 95. (2) Sakamoto, K.; Hirai, K.; Kitamura, Y.; Yamazaki, K.; Yusa, M.; Tokunaga, N.; Doi, G.; Noda, Y.; Tachibana, H.; Segawa, S. Biopolymers 2009, 91, 665. (3) Rohl, C. A.; Baldwin, R. L. Biochemistry 1997, 36, 8435. (4) Konarev, P. V.; Volkov, V. V.; Sokolova, A. V.; Koch, M. H. J.; Svergun, D. I. Journal of Applied Crystallography 2003, 36, 1277. (5) Bernado, P.; Mylonas, E.; Petoukhov, M. V.; Blackledge, M.; Svergun, D. I. Journal of the American Chemical Society 2007, 129, 5656. (6) Zimm, B. H.; Stockmayer, W. H. The Journal of Chemical Physics 1949, 17, 1301. (7) Wishart, D. S.; Sykes, B. D.; Richards, F. M. Biochemistry 1992, 31, 1647. (8) Wishart, D. S.; Sykes, B. D.; Richards, F. M. Journal of Molecular Biology 1991, 222, 311. (9) Krizova, H.; Zidek, L.; Stone, M. J.; Novotny, M. V.; Sklenar, V. Journal of Biomolecular NMR 2004, 28, 369. (10) Tjandra, N.; Grzesiek, S.; Bax, A. Journal of the American Chemical Society 1996, 118, 6264. (11) Bernadó, P.; Blackledge, M.; Sancho, J. Biophysical Journal 2006, 91, 4536. (12) Ortega, A.; Amorós, D.; García de la Torre, J. Biophysical Journal 2011, 101, 892. (13) de la Torre, J. G.; Huertas, M. L.; Carrasco, B. Biophysical Journal 2000, 78, 719.

PAGE S38