Structure-Based Insights into the Dynamics and Function of Two

Article Cite This: Biochemistry XXXX, XXX, XXX−XXX

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Structure-Based Insights into the Dynamics and Function of TwoDomain SlpA from Escherichia coli Anne-Juliane Geitner,† Ulrich Weininger,‡ Hauke Paulsen,§ Jochen Balbach,*,‡,∥ and Michael Kovermann*,‡,∥,⊥ †

Laboratorium für Biochemie, Universität Bayreuth, D-95440 Bayreuth, Germany Institut für Physik, Biophysik, Martin-Luther-Universität Halle-Wittenberg, D-06099 Halle (Saale), Germany § Institut für Physik, Universität Lübeck, Ratzeburger Allee 160, D-23562 Lübeck, Germany ∥ Mitteldeutsches Zentrum für Struktur und Dynamik der Proteine (MZP), Martin-Luther-Universität Halle-Wittenberg, D-06099 Halle (Saale), Germany ⊥ Universität Konstanz, Fachbereich Chemie, Universitätsstraße 10, D-78457 Konstanz, Germany ‡

S Supporting Information *

ABSTRACT: SlpA (SlyD-like protein A) comprises two domains, a FK506 binding domain (FKBP fold) of moderate prolyl cis/trans-isomerase activity and an inserted in flap (IF) domain that hosts its chaperone activity. Here we present the nuclear magnetic resonance (NMR) solution structure of apo Escherichia coli SlpA determined by NMR that mirrors the structural properties seen for various SlyD homologues. Crucial structural differences in side-chain orientation arise for F37, which points directly into the hydrophobic core of the active site. It forms a prominent aromatic stacking with F15, one of the key residues for PPIase activity, thus giving a possible explanation for the inherently low PPIase activity of SlpA. The IF domain reveals the highest stability within the FKBP-IF protein family, most likely arising from an aromatic cluster formed by four phenylalanine residues. Both the thermodynamic stability and the PPIase and chaperone activity let us speculate that SlpA is a backup system for homologous bacterial systems under unfavorable conditions.

S

lyD (sensitive to lysis D) proteins1 belong to the FKBP (FK506 binding protein) family of peptidyl-prolyl cis/transisomerases (PPIases).2 They consist of two separated domains hosting the PPIase2,3 (FKBP domain) as well as chaperone activity3−6 [insert in flap (IF) domain]. SlyD-like or FKBP-IF proteins are structurally4−9 well characterized, and their assumed relationship between intrinsic dynamics and enzymatic activity has been reported in great detail10−12 and recently reviewed.13 A synergistic interplay between both domains of SlyD is suggested to be crucial for increasing the PPIase activity for protein substrates by 2 orders of magnitude compared to that of single domain hFKBP12.3,14 This dynamic cooperation involves a high interaction probability and rapid binding by the IF domain of a broad range of unfolded and aggregation-prone proteins and signal peptides.4,5,10,15 Escherichia coli holds a second FKBP-IF protein, SlpA (SlyDlike protein A), with moderate PPIase activity.2 Recently, Quistgaard et al.16 determined the crystal structure of EcSlpA, confirming the SlyD-like overall fold. Herein, the N-terminal purification tag comprising 11 unfolded residues binds to the IF domain (holo EcSlpA). Additionally, numerous ribosomal proteins were identified as interaction partners of EcSlpA. Moreover, a substantiated chaperone activity of EcSlpA was verified.16 In the work presented here, the structure and dynamics of apo EcSlpA were studied in solution by nuclear magnetic resonance (NMR) spectroscopy and its thermodynamics and potential substrate interaction were elucidated. A detailed © XXXX American Chemical Society

comparison of EcSlpA with various homologues revealed F37 as a potential key residue for the moderate PPIase activity. The IF domain of EcSlpA displays the highest thermodynamic stability of all homologues studied so far most probably because of an unparalleled aromatic cluster formed by F82, F87, F101, and F125. Therefore, this IF domain is well-folded, showing chaperone activity even in the absence of the FKBP domain. We speculate that EcSlpA is a highly stable chaperone backup system for SlyD under hostile cellular conditions.

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MATERIALS AND METHODS Proteins and Peptides. The gene fragments for the SlpA variants were cloned into expression plasmid pET11a (Novagen, Madison, WI) via its NdeI and BamHI restriction sites. The proteins were overproduced in E. coli BL21(DE3)pLysS (Stratagene, La Jolla, CA) as described previously.3 Sitedirected mutagenesis of SlpA was performed via blunt end mutagenesis. 15N-labeled SlpA and 15N- and 13C-labeled SlpA were expressed in M9 minimal medium with [13C]glucose (2 g/L) and/or [15N]NH4Cl (1 g/L) purchased from CortecNet (Voisins-Le-Bretonneeux, France) as the sole carbon and nitrogen sources, respectively. Soluble proteins were obtained after cell lysis with a microfluidizer in 50 mM Tris-HCl, 50 mM NaCl, and 10 mM EDTA (pH 8.0). They were purified on a Received: August 14, 2017 Revised: November 17, 2017 Published: November 20, 2017 A

DOI: 10.1021/acs.biochem.7b00786 Biochemistry XXXX, XXX, XXX−XXX

Article

Biochemistry

were followed for each titration step by 1H−15N HSQC spectra. The overall change in chemical shift values,26 Δδall, was calculated using eq 3:

Ni-NTA column (elution with 250 mM imidazole) and gel filtration on a Superdex 75 prep-grade column (GE Healthcare, Uppsala, Sweden). Apo EcSlpA protein comprises 154 amino acids, including six C-terminal histidines for purification. Expression and purification of the RnaseT1 variant (S54G/ P55N) were performed as previously described.17 The RCM form (reduced and carboxymethylated form of the S54G/P55N double mutant) of RnaseT1 was prepared using the methods described in refs 18 and 19. The tetrapetide Suc-Ala-Leu-ProPhe-pNA (succinyl-Ala-Leu-Pro-Phe-4-nitoanilide) was purchased from Bachem (Bubendorf, Switzerland), whereas the tetrapeptide Abz-ALPF-pNA (aminobenzoyl-Ala-Leu-Pro-Phe4-nitroanilide) was obtained from T. Aumüller (Technical University Munich, Munich, Germany). AEDANS (5-{[(acetylamino)ethyl]amino}naphthalene-1sulfonate)-labeled variants of RNaseT1 [(C2S,C6S,C10N,P39A,S54G,P55N,W59Y)-RNaseT1] have been produced as previously described.20 The concentration of the particular protein solution was determined by the absorbance at a wavelength of 280 nm (ε280 = 1490 M−1 cm−1). All buffers were degassed before being used. NMR Measurements. All NMR spectra were recorded in 100 mM potassium chloride (pH 7.5) containing 10% (v/v) D2O at 298 K using a protein concentration of 1 mM [except for titration experiments (see below)] in a volume of 500 μL. All experiments were performed on a Bruker Avance 800 III spectrometer equipped with a cryoprobe, except 15N R1, R2 relaxation, and amide exchange experiments at pH 7.0, which were performed on a Bruker Avance 600 III instrument equipped with a QXI probe. All spectra were processed using NMRPipe21 and analyzed using NMRView.22 For RDC measurements, EcSlpA was aligned in 18 mg/mL PF1 phages, purchased from PROFOS. Alignment tensors were calculated using Module.23 Hydrogen/deuteron exchange experiments24 were performed at two pH values, 7.0 and 7.5. The experimental dead time was 5 min each. Two-dimensional 1H−15N HSQC spectra were recorded every 20 min (pH 7.5) or 40 min (pH 7.0) at 298 K. The apparent exchange rate constant kex for any detectable cross peak signal was determined using a single-exponential function to the individual volume decay. Protection factor P and thermodynamic stability ΔG were calculated in an amino acid-resolved manner: P=

kch kex

Δδ all =

1 (Δ15N)2 25

2

(3)

where Δ1H is the change in the proton chemical shift and Δ15N is the change in the nitrogen chemical shift. Binding curves of the interaction of the tetrapeptide and RCM-T1 with EcSlpA were fitted to recorded data using eq 4 (B + nc + KD)2 − B − nc − KD + 4nc B 2B c0 − c where B = c0 pep

Δδ all(c) = −a

(4)

where a is the maximum of Δδ , KD is the dissociation constant, n is the stoichiometry, c is the individual tetrapeptide/ RCM-T1 concentration, c0 is the start concentration of EcSlpA, and c0pep is the concentration of the tetrapeptide/RCM-T1 stock solution. One-dimensional 1H spectra for apo EcSlpA were recorded and analyzed according to ref 27 to probe the folding to unfolding transition as induced by temperature. Temperature calibration was performed using standard protocols.28 Structure Calculation. Backbone resonances of 13C- and 15 N-labeled apo EcSlpA were assigned using HNCA,29 HNCACB,30 and HN(CO)CACB31 triple-resonance experiments. Side-chain information was obtained via H(C)CHTOCSY32 and HBHA(CO)NH33 spectra. Three-dimensional (3D) NOESY-edited HSQC experiments34 for 15N and 13C aliphatic/aromatic nuclei confirmed and finalized the side-chain assignment. ARIA 2.335,36 runs were performed using ambiguous NOEs, TALOS37 derived dihedral information,38 and two different alignment tensors based on 1H/15N residual dipolar coupling measurements39 as structural restraints. Ramachandran analysis was performed with PROCHECKNMR.40 The structural ensembles were aligned and illustrated using Pymol 0.99rc6 (DeLano Scientific LLC, 2006). Circular Dichroism (CD) Spectroscopy. Temperatureand urea-induced unfolding of wild-type protein and different EcSlpA variants was monitored at a wavelength λ of 230 nm in 100 mM KP at pH 7.0. Computation. All-atom molecular dynamics (MD) simulations were performed with the GROMACS 4.5.6 software package41−44 using the CHARMM27-CMAP force field45,46 together with the TIP4P water model.47 The lowest-energy structure of EcSlpA obtained via NMR structure calculation was used as the starting protein structure for the MD simulations. A neutral system was reached by adding 77 Na+ and 61 Cl− ions together with 32656 water molecules to the protein. The complete system was placed in a cubic box with periodic boundary conditions and a side length of ∼10 nm. Electrostatic interactions were treated by the particle mesh Ewald (PME) method.48 The lengths of all bonds involving hydrogen atoms were constrained by applying the LINCS algorithm49 for the protein and the SETTLE algorithm50 for water molecules. A time step of 2 fs and an isothermal isobaric (NPT) ensemble were used. Weak coupling to an external temperature and removing the center of mass motion were achieved by velocity rescaling.51 The extended ensemble Parrinello−Rahman all

(1)

where kch is the intrinsic exchange rate constant derived from model peptides25

ΔG = RT ln P

(Δ1H)2 +

(2)

where T is the temperature and R is Boltzmann’s constant. Titration with Proteins and Peptides. For the NMR titration experiments, the tetrapeptide Suc-Ala-Leu-Pro-PhepNA (stock solution; ctetrapeptide = 10 mM) and RCM-T1 (stock solution; cRCM‑T1 = 0.46 mM) were added stepwise up to a 10fold (tetrapeptide) or 2-fold excess (RCM-T1) with regard to 15 N-labeled EcSlpA (start concentration for tetrapeptide titration, cEcSlpA = 0.89 mM; start volume, V = 540 μL; start concentration for RCM-T1 titration, cEcSlpA = 0.22 mM; start volume, V = 470 μL), yielding total final volumes of 1005 μL (for titrating tetrapeptide to EcSlpA) and 895 μL (for titrating RCM-T1 to EcSlpA), respectively. The peak intensity and the chemical shift of each assigned protein cross peak of EcSlpA B


Article

Biochemistry approach52,53 was used to couple the pressure in the simulation box to the atmospheric pressure. A trajectory of 48.1 ns was simulated to analyze the root-mean-square fluctuations (RMSF) of each Cα atom. Peptide Assay for Prolyl Isomerase Activity. The prolyl isomerase activities of SlpA proteins were measured by a protease-free assay as described in ref 54. For the assay, 750 μM peptide substrate Abz-ALPF-pNA was dissolved in anhydrous trifluoroethanol containing 0.55 M LiCl. Under these conditions, approximately 50% of the peptide molecules are in the cis conformation. Upon dilution with 0.1 M potassium phosphate (pH 7.5) to a final peptide concentration of 3 μM, the cis/trans isomerization was initiated and followed by the change in fluorescence at 416 nm (5 nm bandwidth) after excitation at 316 nm (3 nm bandwidth) in 10 mm cells at 288 K. Under these conditions, the cis/trans isomerization of the prolyl bond was a monoexponential process, and its rate constant increased linearly when plotted as a function of the prolyl isomerase concentration. The catalytic efficiency kcat/KM was determined from the slope of this plot. In these experiments, the enzyme concentrations typically varied between 0 and 200 nM. Catalysis of Proline-Limited Protein Refolding. For the protein refolding assays, RCM-T1 was unfolded by incubating 10 μM protein in 0.1 M Tris-HCl (pH 8.0) at 288 K for at least 1 h. Refolding at 288 K was initiated by a 100-fold dilution of the unfolded RCM-T1 to final conditions of 2.0 M NaCl, 0.1 M Tris-HCl (pH 8.0), and the desired concentrations of the prolyl isomerase; 85% of unfolded RCM-T1 molecules fold in a monophasic reaction, which is rate-limited by the slow trans → cis isomerization at Pro39. The remaining 15% of the RCM-T1 molecules contain a correct cis-Pro39 and refold rapidly during the dead time of manual mixing. The folding reaction was followed by measuring the increase in protein fluorescence at 320 nm (5 nm bandwidth) after excitation at 268 nm (3 nm bandwidth). Determination of Chaperone Activity. The chaperone activities of EcSlpA and of the single IF domain of EcSlpA were measured by a citrate synthase aggregation assay as described previously.55 Citrate synthase (c = 30 mM) has been unfolded for 1 h by using 50 mM Tris-HCl (pH 8), 20 mM DTE (dithioerythritol), and 6 M guanidinium chloride (GdmCl). Unfolded citrate synthase aggregates upon 200-fold dilution

were performed in 0.1 M Tris-HCl and 2.0 M NaCl at pH 8.0 and 288 K. All titration curves were analyzed by assuming a 1:1 binding stoichiometry. The kinetics of binding after stoppedflow mixing was measured using a DX.17MV sequential-mixing stopped-flow spectrometer from Applied Photophysics (Leatherhead, U.K.). The path length of the observation chamber was 2 mm, and a 10 mM solution of pNA in ethanol in a 0.2 cm cell was inserted in front of the emission photomultiplier to absorb scattered light and fluorescence below 450 nm from the excitation beam. The kinetics were followed by fluorescence above 450 nm after excitation at 280 nm (10 nm bandwidth). The experiments were performed in 0.1 M Tris-HCl and 2.0 M NaCl at pH 8.0 and 288 K. Kinetic curves were measured 10 times under identical conditions, averaged, and analyzed as monoexponential functions. All titration curves were analyzed by assuming a 1:1 binding stoichiometry. Titration curves and profiles obtained for binding amplitudes were analyzed by assuming a 1:1 binding stoichiometry with F − F0 = (F ∞ − F0) [P]0 + [S]0 + KD −

([P]0 + [S]0 + KD)2 − 4[P]0 [S]0 2[P]0

(5)

where F is the measured fluorescence, F0 and F∞ are the initial and final values, respectively, KD is the dissociation constant for binding, and [P]0 and [S]0 are the protein and substrate concentrations, respectively.

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RESULTS Overall Topology of EcSlpA in Solution. More than 98% of resonances of apo EcSlpA could be assigned by standard triple-resonance NMR experiments (RCSB ID code rcsb103118) for subsequent NMR structure determination. Statistics for the final structure run of apo EcSlpA are listed in Table 1. The solution NMR structure of apo EcSlpA [Protein Data Bank (PDB) entry 2m2a] is presented in Figure 1 and displays a high convergence of the structural ensemble within the FKBP and IF domain (Supplementary Figure 1A,B) but does not show a defined orientation between both domains. This is in good agreement with NMR-based structural studies for homologue proteins like MtFKBP17,8 MjFKBP26,9 EcSlyD,4,7 and HpSlyD6 as well as an alignment of different crystal structures obtained for TtSlyD5 all reporting a loose orientation of the FKBP and IF domain. A superposition of the structure of apo EcSlpA presented here with the crystal structure of holo EcSlpA (Figure 1A,B) disclosed very few differences for both domains, including their active sites. The analysis of 1H−15N residual dipolar couplings (RDCs) yielded two independent alignment tensors for both domains in EcSlpA. Plotting the back-calculated RDC values as obtained from the final structure against the experimentally determined RDCs shows a strong correlation (Supplementary Figure 1C,D). Side-Chain Orientation of Active Site-Forming Residues. Next the side-chain orientation of all residues comprising the active site in the FKBP domain (F15, A25, E26, A35, F37, S43, L44, S45, L48, F71, V134, and F136) was investigated. The orientation of all active site residues is highly refined in the structural ensemble of apo EcSlpA (Figure 1C) except F71. Allatom MD simulations partially confirmed this finding, where the RMSF values for the atoms forming the side-chain of F71 in apo EcSlpA are the highest among all residues comprising the

from 6 M GdmCl buffer in 50 mM Tris-HCl (pH15°C 8.0), which leads to a strong increase in the extent of light scattering. Dilution to a final concentration of 0.15 μM citrate synthase in 50 mM Tris-HCl (pH 8.0), 0.1 mM dithioerythritol, and 30 mM GdmCl is measured in the absence of EcSlpA variants or after addition of various concentrations of EcSlpA or the isolated IF domain of EcSlpA. The stray light intensity has been observed by detecting the intensity at an emission wavelength of 360 nm at a JASCO FP-6300 fluorescence spectrometer at 288 K. The bandwidth for excitation was set to 1 nm; the emission bandwidth was set to 3 nm, and damping was 0.5 s. Equilibrium and Kinetic Measurements of Substrate Association. The binding of 1 μM AED-RNaseT1 [(C2S,C6S,C10N,P39A,S54G,P55N,W59Y)-RNaseT1] to the proteins was followed by the increase in AEDANS fluorescence at a wavelength of 475 nm (5 nm bandwidth) upon the transfer of energy from the Trp in the chaperone domain of the chimeric proteins to AED-RNaseT1 after complex formation. Excitation was at 295 nm (3 nm bandwidth). Measurements C


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Biochemistry Table 1. Statistics for NMR Structure Calculation of Apo SlpA from E. coli experimental restraints ambiguous NOEsa intraresidue (|i − j| = 0) sequential (|i − j| = 1) short-range (|i − j| = 2 or 3) medium-range (|i − j| = 4 or 5) long-range (|i − j| > 5) dihedrals RDCs NOE violations (Å) >0.5 >0.3 >0.1 NOE RMSD energy (kcal/mol) Etot Ebond Eangle Eimproper ENOE Ecdih Esani backbone/heavy atom RMSDb (Å) FKBP domain FKBP secondary structure IF domain IF secondary structure Ramachandran analysisc (%) mostly favored additionally allowed generously allowed disallowed deviations from idealized geometry bond lengths (Å) bond angles (deg) impropers (deg)

3658 1373 1080 605 177 1276 244 51 1.4 ± 0.9 5.9 ± 2.5 93 ± 7 0.041 ± 0.014 740 ± 210 30 ± 2 210 ± 30 120 ± 20 300 ± 170 60 ± 20 20 ± 5 1.2 0.8 1.0 0.8

± ± ± ±

0.3/1.7 0.2/1.2 0.2/1.4 0.2/1.2

± ± ± ±

0.3 0.2 0.2 0.2

84.7 9.9 3.8 1.5 0.0027 0.51 0.80

a

The number of NOE restraints exceeds the number of ambiguous NOEs because of possible multiple interpretations of ambiguous NOEs. bThe RMSD was calculated among 10 refined structures of EcSlpA. cAnalysis by PROCHECK NMR.40

active site (Supplementary Table 1). This is in total agreement with multiple orientations of the corresponding Y68 in the NMR structure of EcSlyD*4 (comprising M1-D165 of EcSlyD) and different orientations of the corresponding Y63 in various crystal structures determined for TtSlyD.5,56 Additionally, the side-chain orientation of F71 as seen in the lowest energy structure of apo EcSlpA differs from the corresponding sidechain orientation of homologue proteins, including holo EcSlpA (Figure 1D), by pointing into the hydrophobic core of the FKBP domain. An alignment based on the secondary structural elements of the FKBP domains of apo EcSlpA with holo EcSlpA (PDB entry 4DT4, crystal structure) as well as EcSlyD* (PDB entry 2K8I, NMR structure), FKBP12 (PDB entry 1FKF, crystal structure), HpSlyD (PDB entry 2KR7, NMR structure), MtFKBP17 (PDB entry 1IX5, NMR structure), MjFKBP26 (PDB entry 3PR9, crystal structure), and TtSlyD (PDB entry 3CGM, crystal structure) illuminates one crucial structural difference. The side-chain orientation of F37 of apo and holo EcSlpA differs strongly from the side-chain orientation of the

Figure 1. Structure of apo EcSlpA and orientation of side-chains belonging to the active site and in comparison to homologue proteins. (A) Alignment of the FKBP domain comprising residues of apo EcSlpA (colored light blue) with the FKBP domain of holo EcSlpA16 (colored marine) and the FKBP domain of EcSlyD*4 (colored olive). All FKBP domains are shown as cartoons. The IF domain for all three proteins is shown as a ribbon. (B) Alignment of the IF domain comprising residues of apo EcSlpA with the IF domain of holo EcSlpA16 and the IF domain of EcSlyD*.4 All IF domains are shown as cartoons. The color coding is the same as in panel A. (C) Side-chain orientation of active site residues of apo EcSlpA within an ensemble comprising the 10 lowest-energy structures as calculated in this study. (D) Three-dimensional orientation of all aromatic side-chains belonging to the active site of apo EcSlpA (colored light blue), holo EcSlpA 16 (colored marine), MtFKBP17 8 (colored orange), MjFKBP269 (colored dark orange), TtSlyD5 (colored bright orange), HpSlyD6 (colored light orange), FKBP1257 (colored yellow-orange), and EcSlyD*4 (colored olive). The assignment of residues corresponds to the sequence of EcSlpA. Note that the side-chain of F37 belonging to EcSlpA is ∼120° tilted (χ1) compared to the corresponding aromatic side-chain orientation of all homologue proteins presented and is shown by a double arrow. In addition, the side-chain orientation of F71 belonging to apo EcSlpA differs significantly from the corresponding aromatic side-chain orientations of all other proteins shown.

corresponding residue of all other structural homologues (Figure 1D). It points strongly into the active site of the FKBP domain where it forms an aromatic stack with F15. It is ∼120° (χ1) tilted compared to the side-chain orientation of the corresponding Y34 of EcSlyD*, F48 of FKBP12, Y29 of TtSlyD, F39 of HpSlyD, F50 of MtFKBP17, and I46 of MjFKBP26, which all show exposed orientations (Figure 1D). It should be noted that the homologue position of F15 in EcSlpA is Y13 in TtSlyD, which forms key interactions with the PPIase inhibitor and transition state analogue FK506.56 For the sake of completeness, the structural alignment of EcSlpA and homologue proteins focusing on the side-chain orientation of all nonaromatic side-chains belonging to the active site of apo D


Article

Biochemistry EcSlpA does not reveal major differences (Supplementary Figure 1E). PPIase, Refolding, and Chaperone Activity of EcSlpA. Probing the PPIase and refolding activity enables us to illuminate the potential impact of structural differences (as presented above) on the function of EcSlpA. Established protocols54 were used to determine the PPIase and refolding activity of apo EcSlpA and six other organisms (see the sequence alignment in Supplementary Figure 2) as well as one chimeric protein composed of the FKBP domain of EcSlyD* and the IF domain of EcSlpA [EcSlyD*ΔIF+IF(EcSlpA)]. The prolyl cis/trans isomerization activity of EcSlpA as probed by using the tetrapeptide Abz-Ala-Leu-Pro-Phe-pNA assay confirms the reported values2 of catalytic efficiency kcat/KM [1.2 × 103 M−1 s−1 (Table 2)]. Refolding of unfolded RCM-T1 in the

shows a catalytic efficiency for RCM-T1 refolding comparable to that of wild-type EcSlyD* and TtSlyD (Table 2), confirming that the poor PPIase activity of EcSlpA is not caused by its IF domain. Note that both single-domain EcSlyD*ΔIF and EcSlpAΔIF do not show any activity in the case of RCM-T1 refolding (Table 2). This indicates that the low PPIase and refolding activity results from the FKBP domain of EcSlpA, in agreement with the structural elucidations of F37 as presented above (Figure 1D). With regard to the chaperone activity, it has been shown that equimolar concentrations of EcSlpA efficiently suppress the aggregation of insulin.16 We have expanded these findings by applying an archetype citrate synthase assay.55 As expected, EcSlpA efficiently reduces the rate of aggregation of citrate synthase (Supplementary Figure 3A), as well. The isolated IF domain does not show a chaperone activity comparable to that of full length EcSlpA (Supplementary Figure 3B). The IF Domain of SlpA Shows a Remarkably High Stability. To further characterize apo EcSlpA, we probed its local thermodynamic stability by two-dimensional heteronuclear 1H−15N NMR hydrogen/deuterium exchange spectroscopy.24 Following the approach of Bai and Englander,25 we find a Gibbs free energy ΔG for the overall thermodynamic stability of ∼18 kJ mol−1 (Figure 2). This approach requires exchange in

Table 2. Catalytic Efficiencies for SlpA Proteins from Different Organisms and Homologue Proteins As Probed by the Isomerization of Tetrapeptide Abz-Ala-Leu-Pro-PhepNA54 and the Assisted Refolding of RCM-T13 at 288 K kcat/KM (M−1 s−1) proteina

isomerization tetrapeptide

EcSlpA KpSlpA EsSlpA VcSlpA YpSlpA Sf SlpA IbSlpA EcSlpAΔIF TtSlyDb TtSlyDΔIFb EcSlyD*c EcSlyD*ΔIFc FKBP12c FKBP12+IF(SlyD)c EcSlyD*ΔIF+IF(EcSlpA)

(1.22 ± 0.02) × 10 (2.7 ± 0.4) × 103 (4.4 ± 0.6) × 103 (1.8 ± 0.2) × 103 (3.4 ± 0.6) × 103 (2.3 ± 0.3) × 103 (0.60 ± 0.05) × 103 not determined not determined not determined 0.3 × 106 0.2 × 106 0.7 × 106 0.7 × 106 (0.95 ± 0.03) × 106

refolding of RCM-T1 3

(1.0 ± 0.2) × (1.2 ± 0.3) × (1.1 ± 0.3) × (0.30 ± 0.03) (1.6 ± 0.4) × (1.0 ± 0.2) × (0.4 ± 0.1) × no activity 0.3 × 106 0.3 × 104 0.8 × 106 no activity 1.5 × 104 2.9 × 106 (0.19 ± 0.01)

103 103 103 × 103 103 103 103

× 106

Figure 2. Thermodynamic stability of apo EcSlpA probed on a residueby-residue level. The local thermodynamic stability for apo EcSlpA was measured by applying the NMR H/D exchange methodology using two conditions: (i) pH 7.0 and B0 = 14.1 T (bar plot) and (ii) pH 7.5 and B0 = 18.8 T (○). Residues comprising the IF domain are indicated by using a background colored light gray.

a

Abbreviations: Kp, Klebsiella pneumoniae; Es, Enterobacter sakazakii; Vb, Vibrio cholerae; Yp, Yersinia pestis; Sf, Shewanella f rigidimarina; Ib, Idiomarina baltica; Tt, Thermus thermophilus. bFrom ref 5. cFrom ref 14.

presence of EcSlpA resulted in a kcat/KM of 1 × 103 M−1 s−1 (Table 2). This value is by far the lowest catalytic efficiency seen for any dual-domain FKBP-IF protein in the RCM-T1 refolding assay reported so far and is even less than that of single-domain FKBP12 (Table 2). Furthermore, the catalytic efficiency seen for EcSlpA-assisted refolding of RCM-T1 is slightly lower than that for the isomerization of the tetrapeptide (Table 2). This holds for all SlpA proteins from different organisms probed in this study and is in clear contrast to the higher catalytic efficiency seen for EcSlyD*-assisted refolding of RCM-T1 compared to that for the isomerization of the tetrapeptide (Table 2). Next we probed whether the FKBP or the IF domain of EcSlpA or their synergetic interplay is responsible for the low PPIase and refolding activity. This interplay has been shown to be crucial for other members of the FKBP-IF protein family.10,11,58 Toward this end, the IF domain of EcSlpA was exchanged against the endogenous IF domain of EcSlyD*, leading to the EcSlyD*ΔIF+IF(EcSlpA) chimera. This chimera

the EX2 regime, which we verified by the pH dependence of the apparent exchange rate constant kex. Note that NMRdetected hydrogen/deuterium exchange spectroscopy is capable of probing the local thermodynamic stability based on the amide proton of each residue. We find that residues of the IF domain (I79, Q80, E86, D89, M99, I113, and I116) sense the same ΔG values as the FKBP domain (section colored gray in Figure 2) in strong contrast to EcSlyD*59 and TtSlyD,5 where the local stability of the IF domain is significantly lower than the overall stability (see Discussion). The thermodynamic stability study of apo EcSlpA was expanded to temperature-dependent unfolding of the wild-type protein and different EcSlpA variants using CD and onedimensional 1H NMR spectroscopy. The NMR data illuminated a single midpoint, Tm, for the temperature-induced unfolding transition of apo EcSlpA of (330.1 ± 0.1) K (Supplementary Figure 4A−C and Table 3), in excellent agreement with the CD spectroscopy-derived value of (329.0 ± E


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Biochemistry Table 3. Thermodynamic Stabilities against Unfolding Induced by Temperature As Probed by NMR and CD Spectroscopy for Apo EcSlpA and Homologue Proteins protein

Tm (K)

ΔHv (kJ mol−1)

NMR

330.1 ± 0.1 329.1 ± 0.1 331.6 ± 0.1 325.1 ± 0.1 316.8 319.2. ± 0.2 327.0 ± 0.1

220 ± 20 245 ± 20 240 ± 10 130 ± 10 not determined 330 ± 10 295 ± 15

EcSlpA EcSlpACD EcSlpAΔIFCD IF (EcSlpA)CD EcSlyD*CDa EcSlyD*DSCb EcSlyD*ΔIFDSCb a

Table 4. Thermodynamic Stabilities against Unfolding Induced by Urea for Apo EcSlpA, Single-Domain Variants of EcSlpA, and Homologue Proteins As Probed by Fluorescence (Fl) or NMR (NMR) Spectroscopy

From ref 3. bFrom ref 54.

0.1) K (Supplementary Figure 5 and Table 3) and the reported value of 327 K for holo EcSlpA.16 Compared to that of EcSlyD*,3,54 the thermodynamic stability of EcSlpA is significantly higher with a higher Tm that is shifted by >10 K (Table 3). To investigate the thermodynamic coupling of the two domains of apo EcSlpA, temperature-induced folding to unfolding transitions were repeated with the isolated FKBP and IF domains. Deleting the sequentially inserted IF domain in EcSlpA changes only marginally Tm and the van’t Hoff enthalpy of unfolding, ΔHv, compared to those of the full length protein (Supplementary Figure 5 and Table 3). Remarkably, the single IF domain of EcSlpA still shows a cooperative folding transition with a Tm of (325.2 ± 0.1) K (Table 3), confirming the high ΔG values reported for residues of the IF domain in full length EcSlpA (Figure 2). Urea-induced unfolding of apo EcSlpA and its isolated domains is in line with these findings (Figure 3 and Table 4)

protein

D1/2 (M)

m (kJ mol−1 M−1)

ΔGU (kJ mol−1)

EcSlpAFl EcSlpAΔIFFl IF (EcSlpA)Fl EcSlyD*ΔIF+IF(EcSlpA)Fl TtSlyDF,a,b TtSlyDΔIFFla,b IF (TtSlyD)Fla,b EcSlyD*Flc EcSlyD*Fld EcSlyD*NMRe EcSlyD*ΔIFFlc EcSlyD*ΔIFFld EcSlyD*ΔIFNMRe FKBP12Flc FKBP12+IF(EcSlyD*)Flc

4.3 3.5 2.7 4.9 3.1 3.7 1 2.6 2.2 2.2 3.2 2.6 2.9 2.6 1.9

6.4 4.4 2.8 6 11.4 8.7 4.3 6.3 6.4 8 4.8 4.6 4.7 7.8 8.1

30f 15.6f 7.7f 29.8f 35.1g 32g 4.3g 16.2h 14.2g 17.7f 15.2h 12.0g 13.6f 19.9h 15.2h

a

GdmCl. bFrom ref 5. cFrom ref 14. dFrom ref 54. eFrom ref 59. fT = 288 K. gT = 298 K. hT = 283 K.

F101, and F125 that has no corresponding pattern either in EcSlyD* (V79, F84, A98, and G121) or in TtSlyD (V74, F79, A93, and F117) (Supplementary Figure 6). Linkage of Thermodynamic Stability and Refolding Activity. Having experimental data for both the thermodynamic stability (D1/2 values in Figure 4A) and RCM-T1

Figure 4. Impact of thermodynamic stability on refolding activity performed by EcSlpA. (A) Midpoint D1/2 for the equilibrium folding to unfolding transition induced by urea for different proteins belonging to the dual-domain FKBP-IF family (filled bars) and single FKBP domains (slightly filled bars): TtSlyD and TtSlyΔDIF (colored blue), EcSlyD* and EcSlyD*ΔIF (colored red), FKBP12+IF(EcSlyD*) and FKBP12 (colored cyan), EcSlpA and EcSlpAΔIF (colored orange), and EcSlyD*ΔIF+IF(EcSlpA) (colored gray). Experimental conditions for obtaining D1/2 are listed in Table 4. (B) Dependence of catalytic efficiency k cat /K M as probed for refolding of RCM-T1 on thermodynamic stability D1/2 for dual-domain FKBP-IF proteins. Color coding as in panel A. The dashed line acts as guide for the eye. Experimental conditions for obtaining kcat/KM and D1/2 are listed in Tables 2 and 4, respectively.

Figure 3. Urea-induced equilibrium unfolding transition of apo EcSlpA (○ ), EcSlpAΔIF (□ ), EcSlyD* ( ●), EcSlyD*ΔIF (■ ), and EcSlyD*ΔIF+IF(EcSlpA) (△) at 288 K as measured by protein fluorescence at 280 nm after excitation at 260 nm (SlpA and SlpAΔIF) and at 304 nm after excitation at 280 nm [SlyD* variants and EcSlyD*ΔIF+IF(EcSlpA)]. The fractional changes as a function of temperature were obtained from two-state analyses of the data (solid lines). The transitions were measured with 4 μM protein in 0.1 M potassium phosphate (pH 7.5) with a path length of 1 cm. Thermodynamic parameters, D1/2, and ΔGU are listed in Table 4.

refolding activity (kcat/KM values in Table 2) for five different FKBP-IF proteins in hand, we performed a correlation analysis of both properties (Figure 4B). As a result, a high catalytic efficiency correlates with an inherently low thermodynamic stability for a FKBP-IF protein. As pointed out above and depicted in Figure 4A, the respective IF domain dictates the stability of the FKBP-IF proteins, although remote from the active PPIase site. The isolated FKBP domains without the IF domains (pale bars in Figure 4A) show a much smaller

showing unfolding midpoints (D1/2) of 4.3 and 3.5 M for EcSlpA and EcSlpAΔIF, respectively. Again, the isolated EcSlpA IF domain shows a remarkably cooperative unfolding with a D1/2 of 2.7 M. The D1/2 = 4.9 M midpoint for the protein chimera EcSlyD*ΔIF+IF(EcSlpA) verifies the extraordinary stabilizing effect of the EcSlpA IF domain also in an EcSlyD* “background”. One structural explanation of this high stability might be a prominent aromatic cluster formed by F82, F87, F


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Biochemistry

performed for 48.1 ns corresponding to ∼3 times the expected rotational correlation time for a protein of this size and topology.10 We speculate that a high plasticity of the IF domain is required for a nonspecific binding of different chaperone substrates; the latter has been reported previously for EcSlyD* using protein substrate libraries.61 A high local flexibility of A42−S45 on that time scale was found in EcSlyD* and TtSlyD and discussed as a dynamic fingerprint for interdomain communication.10 This flexibility is missing in apo EcSlpA and might explain the inherently low PPIase activity of EcSlpA in addition to the stacking interaction observed for F15 and F37. Elucidation of the Substrate Binding Sites in EcSlpA. Two-dimensional 1H−15N NMR spectroscopy was employed to unravel the interaction sites of both the Suc-Ala-Leu-ProPhe-pNA (tetrapeptide), the standard substrate for measuring PPIase activity62 (Table 2), and RCM-T1,18,19 for probing refolding activity toward 15N-labeled EcSlpA. The titration of the tetrapeptide leads to changes in the chemical shifts of EcSlpA according to fast exchange on the NMR time scale (Supplementary Figure 9A). The largest changes in chemical shift values [Δωm > 0.1 ppm (Supplementary Figure 9B)] were observed in both domains of EcSlpA (Figure 6A), nicely representing active sites residues of the FKBP domain and key residues of the substrate binding site in the IF domain16 (Figure 6B). These sites are in good agreement with interacting residues found in earlier studies of EcSlyD*4,10 and TtSlyD.5 The regression of eq 4 to single residue-by-residue profiles leads to a stoichiometry value, n, of approximately 0.5−1 and a dissociation constant, KD, of approximately (0.5 ± 0.1) mM (Supplementary Figure 8C−E) for that interaction. The interaction study of the archetype protein substrate RCM-T1 with 15N-labeled EcSlpA (Supplementary Figure 10A) was performed at intermediate to slow exchange on the NMR time scale. The largest change in chemical shift is significantly smaller [Δωm = 0.05 ppm (Supplementary Figure 10B)] than for interaction of the tetrapeptide with EcSlpA [Δωm = 0.4 ppm (Supplementary Figure 9B)] and for the reported interaction of RCM-α-LA with EcSlyD*4 (Δωm ≈ 0.15 ppm) and TtSlyD5 (Δωm ≈ 0.16 ppm). Note that interaction sites between RCMT1 and EcSlpA are located in both domains (Supplementary Figure 10B). Again, the local interaction sites between RCMT1 and EcSlpA agree very well with residues in the FKBP domain that are responsible for PPIase activity (Figure 6C). Qualitative evaluation of individual titration profiles leads to a stoichiometry value, n, of approximately 0.5 [two EcSlpA molecules per RCM-T1 molecule (Supplementary Figure 10C−E)]. Kinetics of Substrate Binding of EcSlpA. Finally, fluorescence resonance energy transfer (FRET) between a protein substrate (AED-RNase T1) and EcSlpA was utilized to study their association and dissociation kinetics. To function as a FRET acceptor, Y81 of EcSlpA was substituted with tryptophan, and for comparison, the homologue EcSlyD* D101W was designed according to the inverse donor−acceptor pair RCM-RNase T1-P39A/EcSlyD* D101C-AEDANS that has been successfully established previously.63 Detection of the time-dependent fluorescence signal of EcSlpA Y81W and EcSlyD* D101W at varying concentrations of AED-RNaseT1 leads to rate constants of association [kon = 38 ± 1 μM−1 s−1 (EcSlpA Y81W), and kon = 9 ± 1 μM−1 s−1 (EcSlyD* D101W)] as well as rate constants of dissociation [koff = 18 ± 2 s−1 (EcSlpA Y81W) and koff = 10 ± 2 s−1 (EcSlyD* D101W)]

variation in stability. This suggests that the thermodynamic coupling of both domains has functional implications. A final confirmation of this coupling would be determining that destabilizing point mutations in the IF domain of EcSlpA increase its catalytic efficiency, which will be done in the future. Local Dynamics of EcSlpA on a Nanosecond to Picosecond Time Scale. We probed the backbone dynamics of apo EcSlpA on a picosecond to nanosecond time scale by NMR relaxation experiments60 and found lowest backbone hNOE values around A90 and D105 (Figure 5), indicating large

Figure 5. Backbone dynamics of apo EcSlpA on a picosecond to nanosecond time scale. Heteronuclear NOE, hNOE, obtained for apo EcSlpA at 298 K and a B0 of 18.8 T. Residues comprising the IF domain are indicated by using a background colored light gray. Active site residues of the FKBP domain are colored blue, whereas key residues of the substrate binding site in the IF domain are colored red (see Figure 6).

amplitudes of motions of the 1H−15N bond vectors. Most key residues of the substrate binding site in the IF domain16 (F82, E86, F87, A90, L100, T102, M104, and E108 colored red in Figures 5 and 6B) show low backbone hNOE values and

Figure 6. Interaction of the substrate with EcSlpA on a residue-byresidue basis. (A) Residues of EcSlpA experiencing changes in chemical shifts, Δδall (eq 3), of >0.1 ppm upon addition of a 10-fold excess of tetrapeptide are colored cyan. (B) Active site residues responsible for PPIase activity of EcSlpA are colored blue, whereas potential substrate binding sites in the IF domain of EcSlpA as proposed by Quistgaard et al.16 are colored red. (C) Residues of EcSlpA experiencing changes in chemical shifts, Δδall (eq 3), of >0.02 ppm upon addition of a 2-fold excess of RCM-T1 are colored cyan.

corresponding R1 and R2 relaxation rates (Supplementary Figure 7A,B) reporting on high local dynamics. All residues forming the active PPIase site in the FKBP domain are rigid on this time scale (colored blue in Figures 5 and 6B and Supplementary Figure 7A,B) . The same bipartite dynamics was found by an all-atom MD simulation (Supplementary Figure 8 with the same red and blue color coding for the backbone and Supplementary Table 1 for PPIase active site residues) G


Article

Biochemistry

substrate with EcSlpA and to SlyD are similar (Figure 7), we speculate that the reduced available space due to displacement of the side-chain of F37 close to the active site in EcSlpA inherently minimizes the isomerization reaction that subsequently takes place. The results found in solution are in agreement with suggestions derived from the crystal structure16 that a missing plasticity of the binding pocket around residues A35 and L48 reduces the PPIase activity. Along these lines, we have studied the interaction of EcSlpA with permanently unfolded polypeptide chains by applying solution NMR spectroscopy at a residue-by-residue level. Interestingly, spectral changes observed for backbone signals of F37 are strongly pronounced for both the interaction of EcSlpA with the tetrapeptide [ΔωF37 = 0.37 ppm (Supplementary Figure 9B)] and the interaction of EcSlpA with RCM-T1 [ΔωF37 = 0.03 ppm (Supplementary Figure 10B)], making F37 one of the most affected residues upon substrate interaction. Hence, the major structural discrepancy in having a different side-chain orientation of F37 in EcSlpA compared to that of the corresponding residue in homologue TtSlyD and EcSlyD* cannot hold as the single reason for the extremely low PPIase activity seen for EcSlpA. The aforementioned aromatic stacking of F37 with F15 may therefore also intrinsically limit the PPIase activity of EcSlpA, and in addition, the impact of the IF domain and its interplay with the FKBP domain on PPIase activity has to be additionally taken into account as discussed below. Mechanistic Implications of the Very Stable IF Domain in EcSlpA. The IF domain of EcSlpA has, to the best of our knowledge, the highest thermodynamic stability of all IF domains reported so far. It is folded and active as a molecular chaperone in isolation and stabilizes FKBP domains (Table 4) when inserted in wild-type proteins (EcSlpA) and chimera (EcSlyD*ΔIF)+IF(EcSlpA). This confirms earlier findings about the thermodynamic coupling of the two domains. The IF domains of both EcSlyD* and TtSlyD are intrinsically less stable and therefore destabilize the FKBP domains in EcSlyD*, TtSlyD, and FKBP12 (Table 4) because the N- and C-termini of the IF domain must be tied together by the FKBP domain.14 In this respect, the IF domain of EcSlpA can function as an artificially introduced disulfide bond, which can stabilize two FKBP12 chimera.58 What is then the structural reason for this high stability of the EcSlpA IF domain? We have identified a prominent aromatic cluster formed by the four phenylalanines, F82, F87, F101, and F125 (Supplementary Figure 6), that has no corresponding pattern in EcSlyD* (V79, F84, A98, and G121) or TtSlyD (V74, F79, A93, and F117). Such aromatic clusters are known to indeed tune the protein stability, e.g., by comparing mesophilic and thermophilic homologues64 or in other very small proteins such as the villin headpiece subdomain,65 where F47, F51, and F58 form the hydrophobic core domain. Further examples include the β-barrel protein CRABP1,66 where phenylalanine−phenylalanine interactions are the key for the overall stability. A high thermodynamic stability might be counterproductive for the enzymatic activity because of the reduced conformational plasticity of the active site. Such a relation emerges when plotting kcat/KM values of different FKBP-IF combinations over stability (Figure 4). In structural terms, the stacking interaction of F37 and F15 blocking the PPIase activity in the FKBP domain cannot be overcome by a destabilizing effect of the IF domain. Along these lines, the dynamic coupling of both domains in EcSlyD* and TtSlyD reflected in a high local backbone dynamics of A42−S4510 is missing in EcSlpA.

(Figure 7A). A subsequent independent analysis of changes in fluorescence amplitude confirms the quantity of binding

Figure 7. Formation of the complex of AED-RNaseT1 with EcSlpA Y81W (●) and EcSlyD* D101W (▲). (A) Dependence of the measured rate constants and (B) dependence of the amplitudes of complex formation on the concentrations of EcSlpA Y81W and EcSlyD* D101W, respectively. Values for koff and kon were derived from the intercept and the slope of the lines in panel A. For EcSlpA Y81W, kon = 38 ± 1 μM−1 s−1 and koff = 18 ± 2 s−1; for EcSlyD* D101W, kon = 9 ± 1 μM−1 s−1 and koff = 10 ± 2 s−1. All data were analyzed assuming a simple 1:1 binding reaction. Lines in panel B represent the analysis by a simple 1:1 binding reaction (see eq 5), yielding dissociation constants (KD) for EcSlpA Y81W of 0.56 ± 0.03 μM and for EcSlyD* D101W of 2.6 ± 0.2 μM. The kinetics were followed by fluorescence above 450 nm after excitation at 280 nm. All measurements were performed in 0.1 M Tris-HCl (pH 8.0) and 2.0 M NaCl at 288 K with 0.5 μM AED-RNaseT1.

kinetics by a dissociation constants (KD) of 0.56 ± 0.03 μM (EcSlpA Y81W) and 2.6 ± 0.2 μM (EcSlyD* D101W) (Figure 7B). The kinetic rate constants for both the association and the dissociation of a protein substrate with EcSlpA are conserved compared to those of homologue FKBP-IF proteins EcSlyD* and TtSlyD4,5,63 because their values are similar and contribute for this reason to the two-step mechanism for acting as a twodomain PPIase as proposed previously:11 first fast binding of the substrate to the IF domain and second interaction of the IFbound complex with the active site in the FKBP domain.

■

DISCUSSION Side-Chain Orientation of Active Site F37 Could Explain the Reduced Isomerase Activity of EcSlpA. The three-dimensional structure of apo EcSlpA presented here confirms that the overall findings of the crystal structure of the N-terminally tagged protein in complex with a substrate in the IF domain16 represent very well the structure in solution. A closer look into the side-chain orientation of active site-forming residues brings F37 into the focus. The aromatic ring of F37 is ∼120° tilted (χ1) compared to the orientation of EcSlyD* homologue position Y34 and all other FKBP-IF proteins for which structures have been determined (Figure 1C). Note that the embedding of F37 into the hydrophobic core of EcSlpA is not compensated by extraordinary fluctuations and dynamics as shown by the all-atom MD simulations (Supplementary Figure 8 and Supplementary Table 1) and hNOE experiments (Figure 5). Instead, F37 forms an aromatic stack with F15, whose homologue Y13 in TtSlyD shows key interactions with the PPIase inhibitor and transition state analogue FK506.56 The accessibility of the hydrophobic core of the PPIase active site is for these reasons significantly reduced compared to those of other FKBP-IF proteins because the side-chain orientation of F71 is still comparable to that of Y68 of EcSlyD*, which is also known as a sterical hindrance for the entry of substrates.4 As, on the other hand, the rate constants for association of the H


Biochemistry

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High Rates of Chaperone−Substrate Association. AED-RNaseT1 is a permanently unfolded protein substrate for EcSlpA, which was used to determine the association and dissociation rate constants by FRET. As found for other FKBPIF domain pairs,20,58 both rate constants are high (kon = 38 μM−1 s−1, and koff = 18 s−1), which facilitates rapid binding of the substrate to the chaperone domain and a short residence time to avoid blocking of the binding site for other substrates. NMR titration with EcSlpA substrates revealed the expected IF domain binding sites (Figure 6) together indicating the same mechanistic progression found for SlyD homologues.4,5,10 The IF chaperone domain attracts aggregation-prone substrates in the proximity of the PPIase active site with a short residence time to release the polypeptide chain for prolyl isomerization if needed. Because of the low PPIase activity of EcSlpA, the latter is a less likely scenario. We propose that EcSlpA mainly functions as a molecular chaperone with a substrate specificity similar to that of homologue SlyD even under harsh cellular conditions because of its high thermodynamic stability, requiring functional adaptation.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.7b00786. Ten figures containing structural alignment using residues comprising the FKBP or IF domain, correlation plots for RDC analysis, sequence alignment, citrate synthase assay, folding to unfolding transition of EcSlpA as monitored by NMR, thermal stabilities of EcSlpA variants as monitored by fluorescence, structural analysis of the hydrophobic cluster seen in the IF domain, 15N relaxation data, root-mean-square fluctuations (RMSF) obtained by using a MD simulation, analysis of tetrapeptide−EcSlpA interaction, and analysis of RCMT1−EcSlpA interaction and one table presenting all-atom RMSF values for residues forming the active site in EcSlpA. (PDF)

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Article

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Ulrich Weininger: 0000-0003-0841-8332 Michael Kovermann: 0000-0002-3357-9843 Funding

This work has been supported by grants from the DFG (GRK 1026, BA 1821/4-1, WE 5587/1), the state Sachsen-Anhalt (Exzellenznetzwerk Biowissenschaften), and ERDF by the EU. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Christian Scholz for providing the six SlpA Proteins from Klebsiella pneumoniae, Enterobacter sakazakii, Yersinia pestis, Vibrio cholerae, Shewanella frigidimarina, and Idiomarina baltica and Dominique Sydow for assisting in MD simulation. I


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