V67L Mutation Fills an Internal Cavity To Stabilize RecA - American

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V67L Mutation Fills Internal Cavity to Stabilize RecA Mtu Intein Allison Sophie Zwarycz, Martin J. Fossat, Otar Akanyeti, Zhongqian Lin, David Jacob Rosenman, Angel E Garcia, Catherine Ann Royer, Kenneth V. Mills, and Chunyu Wang Biochemistry, Just Accepted Manuscript • Publication Date (Web): 10 May 2017 Downloaded from http://pubs.acs.org on May 12, 2017

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Biochemistry

V67L Mutation Fills Internal Cavity to Stabilize RecA Mtu Intein Allison S. Zwarycza, Martin Fossata, Otar Akanyetib, Zhongqian Lina, David J. Rosenmana, Angel Garciac, Catherine A. Royera, Kenneth V. Millsd, Chunyu Wang*a a

Department of Biological Sciences, Rensselaer Polytechnic Institute, Troy, NY, 12180;

b

c

Department of Computer Science, Aberystwyth University, Ceredigion, SY23 3FL, Wales;

Center of Nonlinear Studies, Los Alamos National Laboratory, Los Alamos, NM, 87545;

d

Department of Chemistry, College of the Holy Cross, Worcester, MA, 01610

KEYWORDS. intein, protein splicing, high pressure NMR, protein stability, cavity, volume change of folding

ACS Paragon Plus Environment

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ABSTRACT. Inteins mediate protein splicing, which has found extensive applications in protein science and biotechnology. In the Mycobacterium tuberculosis (Mtu) RecA mini-mini intein (∆∆Ihh), a single valine to leucine substitution at residue 67 (V67L) dramatically increases intein stability and activity. However, crystal structures show that the V67L mutation causes minimal structural rearrangements, with an RMSD of 0.2 Å between ∆∆Ihh-V67 and ∆∆Ihh-L67. Thus, the structural mechanisms for V67L stabilization and activation remain poorly understood. In this study, we used intrinsic tryptophan fluorescence, high-pressure NMR, and MD simulations to probe the structural basis of V67L stabilization of the intein fold. Guanidine (GdnHCl) denaturation monitored by fluorescence yielded a free energy change (∆Gfo) of -4.4 kcal mol-1 and -6.9 kcal mol-1 for ∆∆Ihh-V67 and ∆∆Ihh-L67, respectively. High-pressure NMR showed that ∆∆Ihh-L67 is more resistant to pressure-induced unfolding than ∆∆Ihh-V67. The volume change of folding (∆Vf) was significantly higher for V67 (71 ± 2 mL mol-1) than for L67 (58 ± 3 mL mol-1) inteins. The measured difference in ∆Vf (13 ± 3 mL mol-1) roughly corresponds to the volume of the additional methylene group for Leu, supporting the notion that the V67L mutation fills a nearby cavity to enhance intein stability. In addition, we carried out MD simulations to show that V67L decreases side chain dynamics and conformational entropy at the active site. It is plausible that changes in cavities in V67L can also mediate allosteric effects to change active site dynamics and enhance intein activity.


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Intein-mediated protein splicing is a post-translational and self-catalyzed process in which an intein excises itself from flanking polypeptides, or exteins, with concurrent ligation of the flanking exteins1,2 (For reviews, see Paulus3, Shah and Muir4, or Mills, Johnson, and Perler5). Inteins have found wide applications in protein engineering, labeling, immobilization, purification and precise control of protein function6–9. The Mycobacterium tuberculosis (Mtu) RecA intein is composed of 440 residues, with a splicing domain and a homing endonuclease domain10. It was first minimized to 168 residues through the removal of the endonuclease domain11, and then to 139 residues by the replacement of a long, flexible loop with the residues of a β-turn from the homologous Hedgehog (hh) Hint (Hedgehog-intein) domain12. This “mini-mini” intein (∆∆Ihh) has enhanced stability and splicing activity with an additional V67L substitution within its hydrophobic core12. The V67L mutation also enhances intein activity in many Mtu RecA intein variants12. The activating effect of V67L was independently observed in the directed evolution of the Mtu RecA intein by Liu and coworkers13. In the presence of sodium dodecyl sulfate (SDS), V67L is more resistant to thermolysin digestion12, indicating enhanced structural stability. Using solution NMR, we previously have shown that the V67L mutation decreases hydrogen-deuterium exchange rates globally, consistent with the stabilizing effect of this mutation14. However, the crystal structures of ∆∆Ihh-V67 and ∆∆Ihh-L67 inteins have an RMSD of 0.2 Å, revealing minimal conformational difference15. Thus, the underlying structural mechanism for the enhanced stabilization and activity of V67L is still poorly understood.


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A

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B

V67

L67

Figure 1. ∆∆Ihh intein structures with cavities colored in orange. (a) ∆∆Ihh-V67 (PDB 2IN9). (b) ∆∆Ihh-L67 (PDB 2IN0). Residue 67 is colored magenta in ∆∆Ihh-V67 and blue in ∆∆Ihh-L67. Both structures were rendered using PyMOL version 1.8, with cavities calculated with the program Hollow16.

A possible mechanism for V67L stabilization is filling a nearby cavity by the additional methylene group of the L67 side chain. Imperfect packing in folded proteins creates internal cavities, primarily surrounded by hydrophobic side chains. It has been shown through crystallography, UV absorbance, fluorescence and NMR that mutations that increase cavity size can decrease stability of the protein fold17–20 while smaller cavities can lead to higher stability21,22. In addition, filling the cavity and increasing the density of the protein interior can increase dispersion interactions among non-bonded atoms to increase stability23. Although cavity shapes are visibly different in ∆∆Ihh-L67 and ∆∆Ihh-V67 (Fig. 1) crystal structures, the cavity sizes in solution cannot be accurately assessed from crystal structures alone, because of the dynamics and ensemble behavior of protein cavities in solution. These considerations motivated us to carry out solution NMR studies of ∆∆Ihh-V67 and ∆∆Ihh-L67 at high pressure in order to quantify the volume change of folding. Internal cavities are a major driving force for pressure-


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induced unfolding of proteins24. High pressure (HP) operates on protein volume and favors protein states with smaller volume. Because the unfolded state has smaller volume than the folded state, HP generally unfolds proteins. Pressure-induced unfolding is governed by the volume change of protein folding (∆Vf > 0). Protein internal cavities increase the volume difference between the folded state and the unfolded state, increasing the positive ∆Vf and promoting pressure-induced unfolding. In contrast, volume reduction or elimination of internal cavities can decrease ∆Vf, which, in turn, increases the pressure stability of proteins. In this work, we used high-pressure solution NMR in combination with intrinsic tryptophan fluorescence denaturation and MD simulations to investigate the structural, dynamic and energetic consequences of the V67L mutation. Using these methods, we were able to determine a reduction in cavity volume of 13 ± 3 mL mol-1 by the V67L mutation, validating the cavityfilling mechanism.

MATERIAL AND METHODS Protein overexpression and purification The NMR samples of ∆∆Ihh-V67 and ∆∆Ihh-L67 were prepared as described previously14,25. Briefly, JM101 cells, transformed with the overexpressing plasmid encoding a CBD (chitinbinding domain)-intein fusion protein, were grown at 37°C to OD600 of 0.4, followed by induction with 1 mM IPTG (isopropyl β-D-1-thiogalactopyranoside) and growth for 16-20 hours at 20°C. Isotopic labeling was achieved by using M9 media with 1 g/L

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NH4Cl. The fusion

protein was bound batch-wise to chitin-beads at 4°C, followed by DTT-induced (200 mM dithiothreitol) cleavage of the intein from the CBD fusion protein and size-exclusion chromatography. The pure protein was concentrated and exchanged by ultrafiltration with


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Amicon Ultra centrifugal devices into 20 mM sodium phosphate (pH 7), 100 mM NaCl, and 1 mM NaN3.

Denaturation by guanidine monitored by Trp fluorescence Denaturation experiments were carried out on a Tecan Infinite M1000 Pro plate reader and were performed in triplicate at 25°C, with excitation at 295 nm and emission between 315-400 nm with a measurement bandwidth of 5 nm. Protein concentration was 25 µM in the appropriate denaturant concentration. Samples were in the denaturant for 90 minutes prior to fluorescence measurements to achieve equilibrium.

High-pressure NMR Uniformly

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N-labeled samples were concentrated to 0.55 mM (∆∆Ihh-V67) and 0.50 mM

(∆∆Ihh-L67) in 20 mM Tris (pH 7), 100 mM NaCl, 1 mM NaN3, and 10% D2O, with 2.5 M GdnHCl (∆∆Ihh-V67) or 3.25 M GdnHCl (∆∆Ihh-L67). ∆∆Ihh-L67 did not unfold at high pressure at lower GdnHCl concentration. Experiments were carried out in a high-pressure ceramic NMR tube using an automatic pressure pump (Daedalus Innovations, Philadelphia, PA). Eleven pressure increments, 250 bar each, were performed from 1 bar up to 2500 bar. At each pressure, equilibrium was rapidly reached as evidenced by the lack of change in repeated 1D and 2D NMR spectra. 15N—1H HSQC (Heteronuclear Single Quantum Coherence) experiments were performed at each pressure with 8 and 16 scans for ∆∆Ihh-V67 and ∆∆Ihh-L67, respectively. ∆∆Ihh-L67 required more scans due to the slightly lower protein concentration and higher denaturant concentration. All NMR experiments were carried out at 25°C on a Bruker 600 MHz spectrometer.


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NMR spectra were processed with nmrPipe software26 and analyzed using Sparky (Goddard and Kneller, SPARKY 3, University of California, San Francisco). The backbone HSQC assignment was performed previously for both ∆∆Ihh-L6725 and ∆∆Ihh-V6714. For both ∆∆IhhV67 and ∆∆Ihh-L67, we were able to assign backbone amides for 133 of the 139 residues prior to adding GdnHCl. Assignments in the presence of GdnHCl were obtained by a GdnHCl titration. As GdnHCl concentration increased in the sample, peaks in the HSQCs moved in a straight line. Therefore, the assignment at higher concentration of GdnHCl could be obtained by following the peak movement with increasing concentration of GdnHCl.

Data analysis Data analysis and statistical tests were performed using in-house scripts in Matlab version 2010a (Mathworks, Natick, MA). We first modeled the folding equilibrium as a function of high-pressure. For each residue, we used a sigmoid function to determine the relationship between NMR peak intensity (I) and pressure (p) using the following equation:

=

∆ °[] ∆ ∙ ∆ °[] ∆ ∙

(1)

where R is the universal gas constant; T is temperature in Kelvin; ∆Gfo[D] is the Gibb’s free energy change of folding of the protein at ambient pressure at denaturant concentration [D]; p is pressure; If is NMR peak intensity of resonances corresponding to the folded state; and ∆Vf is the change in volume upon folding. We estimated the parameters of equation 1 (∆Gfo[D] and ∆Vf) using a least square algorithm by minimizing the error between the actual and predicted I(p). To determine whether there were differences in ∆Vf or ∆Gfo[D] between ∆∆Ihh-V67 and ∆∆Ihh-L67,


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we performed a Student’s (unpaired) T-test on residues with: R2 greater than 0.90 in curvefitting, and peaks with high signal-to-noise ratios, no line-broadening, and no overlapping. To extrapolate Gibb’s free energy of folding at zero denaturant (∆GH2O) we used the following equation: ° ∆[] = ∆ +

∙ [!]

(2)

where m is susceptibility to denaturant (i.e. m-value). For high pressure NMR experiments all values shown are mean ± standard error of the mean of the 41 residues chosen. The fluorescence data were fit to the following equation:

"# [!] =

$% $

∆ ° &∙[]

∆ ° &∙[]

(3)

where Cm is the center of mass of the Trp fluorescence scan; ∆Gfo is the Gibb’s free energy of folding; [D] is the concentration of the denaturant; Cu and Cf are the center of mass of the completely unfolded and folded states, respectively. Cu and Cf were fit to be plateau values of Cm at m=0 and ∞, respectively. We estimated the parameters of equation 3 (∆Gfo and m) using a least square algorithm by minimizing the error between the actual and predicted Cm. The center of spectral mass wavelength was calculated using the following equation:

"# =

∑( # ( ) ( ∑( # (

(4)

where mi is the fluorescence intensity at wavelength λi, with the wavelength ranging from 315 nm to 400 nm in 1 nm increments.

Molecular dynamics simulations


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Simulations were performed on ∆∆Ihh-V67 and ∆∆Ihh-L67 using PDB structures 2IN9 and 2IN015, respectively, using the GROMACS 4.5.3 package27. For each structure, the AMBER99sb-ILDN28 force field and the TIP4P-Ew29 water model were used. The PDB structures were protonated to standard pKa values at pH 7.0. To match pKa values determined by an NMR titration30, all histidines were protonated as HIE, except for histidine 138 (protonated as HID) and histidine 128 (protonated as HIP). Each structure was then solvated in a 5.8 nm cubic box, followed by water replacement with ions to 0.03 M NaCl. The resulting simulations included 6,210 waters, 5 Na+ ions, and 4 Cl- ions. To ensure minimal changes in box size, a 1 ns NPT equilibrium simulation, with an integration step of 1 fs, was run at 1 bar and 25°C. Pressure and temperature were coupled using the standard Berendsen method31. Standard NPT MD simulations were performed in triplicate for each structure, with a simulation time of 100 ns for each run. The LINCS32 and SETTLE33 algorithms were used to constrain bond lengths, which allowed for a 2 fs timestep. Electrostatics were calculated used the particle mesh Ewald method34 and Lennard-Jones interactions used a distance cutoff of 1 nm. The Nose-Hoover algorithm35 was used to couple temperature to 25°C and the ParrinelloRahman barostat36 was used to couple pressure to 1 bar. The runs were performed on a Linux cluster at Rensselaer Polytechnic Institute. Analysis was performed on 10-100 ns for each run, as the first 10 ns were determined to be an equilibration period, which was assessed by RMSD graphs vs simulation time compared to the original structures.


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RESULTS AND DISCUSSION

Table 1. ∆Gf and ∆Vf of ∆∆Ihh-V67 and ∆∆Ihh-L67 Experiment/Parameter GdnHCl Fluorescence

High-Pressure NMR (mean and SE over 41 residues)

m-value (L kcal mol-2) ∆Gfo (kcal mol-1) ∆Vf (mL mol-1) ∆Gfo[D] in GdnHCl (kcal mol-1) Extrapolated ∆GH2O (kcal mol-1)

∆∆Ihh-V67

∆∆Ihh-L67

1.25

1.78

-4.4

-6.9

71 ± 2

58 ± 3

-2.4 ± 0.1 (in 2.5 M GdnHCl)

-2.9 ± 0.2 (in 3.25 M GdnHCl)

-5.5 ± 0.1

-8.7 ± 0.2

V67L mutation increases stability against guanidine denaturation unfolding We examined the effect of the V67L mutation on denaturation by urea or guanidine denaturation using intrinsic tryptophan fluorescence. With increasing concentration of GdnHCl, the center of mass of the fluorescence spectrum shifted toward longer wavelengths, consistent with solvent exposure and quenching of tryptophan fluorescence (Fig. 2). The GdnHCl denaturation for both ∆∆Ihh-V67 and ∆∆Ihh-L67 inteins resulted in a sigmoidal curve, indicating overall two-state unfolding. However, the onset of the increase in ∆∆Ihh-V67’s center of mass was at 2.5 M GdnHCl, while ∆∆Ihh-L67 only began to increase at 3.25 M, demonstrating the stabilizing effect of the V67L mutation. The data were fit to equation (3) and, as expected, the ∆Gfo is more favorable for ∆∆Ihh-L67 (-6.9 kcal mol-1) than ∆∆Ihh-V67 (-4.4 kcal mol-1).


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Figure 2. Fluorescence monitoring of GdnHCl denaturation of ∆∆Ihh-V67 and ∆∆Ihh-L67 inteins, with center of mass shift of the fluorescence spectrum as a function of denaturant concentration. ∆∆Ihh-V67 began to unfold at 2.5 M, whereas ∆∆Ihh-L67 started to unfold at 3.25 M.

We also studied the effect of urea on ∆∆Ihh folding. Surprisingly, little change in the fluorescence spectrum was observed, even in the presence of 8 M urea (data not shown). This is consistent with a previous observation that V67L did not affect thermolysin digestion in the presence of urea12. GdnHCl and urea have distinct unfolding mechanisms, with different interactions with protein and solvents, because GdnHCl is charged while urea is neutral. For example, GdnHCl can disrupt native electrostatic interactions in proteins in addition to hydrogen bonds, while urea has little effect on native salt bridges37. The inability of urea to unfold the intein suggests that there are native electrostatic interactions in the intein that are resistant to urea and crucial to the stability of the intein fold. It is also possible that the hydrophobic and hydrogen bonding interactions between GdnHCl and the intein contribute to the unfolding mechanism here.


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High-pressure NMR revealed that V67L decreases ∆Vf by 13 mL mol-1 We hypothesized that the V67L mutation fills a cavity to stabilize the intein fold, which should lead to a smaller value for the ∆V of folding for the V67L mutant with respect to WT protein. This prediction can be directly tested by high-pressure NMR. In order to observe unfolding in the pressure range of the high-pressure NMR apparatus (up to 2.5 kbar), non-denaturing concentrations of GdnHCl were required, 3.25 M and 2.5 M GdnHCl for ∆∆Ihh-L67 and ∆∆Ihh-V67, respectively. Fig. 3A shows the overlay of

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N-1H HSQC

spectra at eleven different pressures, ranging from 1 bar to 2.5 kbar, for ∆∆Ihh-V67 in the presence of 2.5 M GdnHCl. As the pressure increased, most peaks corresponding to the folded conformation in the HSQC showed pressure-dependent movements with concomitant peak intensity decrease. For ∆∆Ihh-V67, peak intensity for the folded conformation was very weak at pressures greater than 1.5 kbar (Figure 3A, light blue), while a new set of peaks, corresponding to the unfolded conformation, became increasingly noticeable, clustering around the amide chemical shift of ~8.3 ppm, typical for disordered conformations. As pressure was increased further to 2.5 kbar, peaks for the folded conformation almost completely disappeared while peaks for the unfolded conformation dominated the spectrum (Figure 3A, dark purple). For ∆∆Ihh-L67, a similar pattern of peak movement and intensity change were observed, except that the protein unfolded at higher pressure, even in the presence of a higher concentration of GdnHCl (3.25 M v. 2.5 M), indicating more resistance to pressure-induced unfolding. We ensured that the system reached equilibrium at each pressure by monitoring the time dependence of the NMR spectrum. The pressure-induced unfolding of the inteins is fully reversible; the HSQC spectra reverted to that of the folded conformation upon return to ambient pressure after the high-pressure NMR experiments were completed.


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Biochemistry

Figure 3. V67L mutation increases resistance to pressure-induced unfolding and decreases the ∆Vf by 13 ± 3 mL mol-1. (a) Overlay of 15N-1H HSQC spectra for ∆∆Ihh-V67 at eleven pressures. As the pressure increased, peak intensity corresponding to folded conformation decreased and new peaks corresponding to unfolded protein (dark purple) began to appear. ∆∆Ihh-L67 showed similar trends, except at higher pressures. 41 labeled peaks, with high signal-to-noise ratios and good resolution at all pressures in both mutants, were used in data fitting. (b) Curve fit with Eq. 1 of four selected residues for ∆∆Ihh-V67 (red) and ∆∆Ihh-L67 (blue). (c) Histogram of ∆Vf calculated from Eq. 1 for the 41 residues; ∆∆Ihh-V67 (red), ∆∆Ihh-L67 (blue), and overlapping region (purple).


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The pressure-dependent decreases in the folded state peak intensity were analyzed to obtain residue-specific values for the free energy and volume change of folding using equation (1). The representative fits for four residues (I61, A84, L87, and V137) (Fig. 3B) revealed a clear shift towards higher pressure for the ∆∆Ihh-L67 variant compared to ∆∆Ihh-V67. These residues line the largest cavity (Fig. 1), which contains the V67L mutation. The values of the residue-specific volume change of folding of ∆∆Ihh-V67 vs ∆∆Ihh-L67 were compared for 41 residues in both ∆∆Ihh-V67 and ∆∆Ihh-L67 variants. The 41 residues were selected based on high signal-tonoise ratio, R2 > 0.90, no line-broadening due to pressure, and no peak overlap. The average volume change of folding was found to be 71 ± 2 mL mol-1 for ∆∆Ihh-V67 and 58 ± 3 mL mol-1 for ∆∆Ihh-L67 (p-value < 0.05) (Table 1, Fig. 3C). The wide distribution of ∆Vf values is likely due to deviations from the two-state folding behavior, e.g. local unfolding and multi-step unfolding. The V67L mutation causes a 13 ± 3 mL mol-1 decrease in the ∆Vf, roughly the size of a methylene group38. This directly supports our hypothesis that the V67L mutation stabilizes the intein fold by filling a nearby cavity. In a previous high-pressure NMR study of the c-Myb R2 subdomain, a V103L mutation was found to have increased folding stability against pressure39, although the same mutation decreased DNA binding activity of this protein.


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Table 2. Change in volume of folding and Gibb’s free energy of folding for 41 residues. Res. G5 T6 T13 E20 D21 V22 G24 G25 R26 V31 D37 T39 H41 S47 G52 I57 G58 I61 A62 I66 T77

∆∆Ihh-V67 ∆Vf ∆Gfo (kcal R2 (mL -1 mol-1) mol ) 70 -2.2 0.98 53 -1.7 0.94 67 -2.4 0.98 64 -2.0 1.00 94 -3.4 0.98 55 -1.5 0.99 64 -2.2 0.99 88 -3.3 0.98 80 -2.8 0.98 68 -2.3 1.00 54 -2.0 0.96 88 -3.2 0.98 52 -1.7 0.98 74 -2.4 0.99 76 -2.3 0.98 109 -4.9 0.96 57 -1.8 0.98 69 -2.4 0.98 59 -1.9 0.98 72 -2.7 0.99 85 -3.0 0.99

∆∆Ihh-L67 ∆Vf ∆Gfo (mL (kcal R2 -1 -1 mol ) mol ) 67 -3.3 0.91 67 -3.4 0.96 63 -3.3 0.94 71 -3.7 0.97 63 -3.1 0.98 36 -1.3 0.94 52 -2.6 0.92 66 -3.4 0.96 68 -3.6 0.98 47 -2.2 0.98 41 -2.0 0.98 54 -2.6 0.97 52 -2.5 0.97 55 -2.8 0.94 41 -1.8 0.94 126 -6.8 0.92 58 -3.0 0.97 64 -3.2 0.96 69 -3.6 0.96 49 -2.4 0.98 82 -4.2 0.97

Res. G80 A84 E86 L87 K89 V93 D97 T100 G101 E102 S106 L112 T114 R115 T119 F120 L130 A132 G134 V137

∆∆Ihh-V67 ∆Vf ∆Gfo (kcal R2 (mL -1 -1 mol ) mol ) 72 -2.7 0.99 61 -1.8 0.98 76 -2.4 0.99 67 -2.1 1.00 111 -3.5 0.99 56 -1.8 0.97 60 -2.1 1.00 75 -2.6 1.00 68 -2.4 0.99 67 -2.2 0.99 64 -2.0 1.00 61 -1.8 0.99 69 -2.3 0.99 59 -1.9 0.99 70 -2.1 0.97 64 -2.0 0.99 121 -4.8 0.99 64 -2.1 1.00 71 -2.1 0.99 67 -2.1 0.99

∆∆Ihh-L67 ∆Vf ∆Gfo (kcal R2 (mL -1 -1 mol ) mol ) 74 -3.8 0.98 47 -2.5 0.91 64 -3.3 0.98 42 -2.0 0.96 35 -1.4 0.95 32 -1.2 0.94 42 -2.0 0.96 60 -3.1 0.95 43 -2.1 0.97 64 -3.2 0.97 63 -3.2 0.97 47 -2.1 0.94 54 -2.7 0.99 45 -2.3 0.98 82 -4.1 0.94 41 -2.2 0.96 79 -3.7 0.91 68 -3.5 0.94 44 -1.7 0.98 69 -3.4 0.95

MD simulations indicate V67L leads to differential distribution of side chain conformation at the active site To further probe the mechanism of V67L activation of the intein, we carried out three 100 ns MD simulations of both V67 & L67 varian of the intein (Fig. 4), with a total simulation time of 600 ns. As expected from the almost identical crystal structures, the contact maps of V67 and L67 inteins from MD are nearly identical (Figure 4A). However, ∆∆Ihh-V67 has higher sidechain flexibility at the active site than ∆∆Ihh-L67, demonstrated by the sampling of sidechain dihedral angles (Figure 4B). In ∆∆Ihh-L67, the catalytic residues C1, D121, and N13930,42 have sidechains mostly sampling a single χ1 angle. However, in ∆∆Ihh-V67 these same side chains sample two χ1 angles with roughly equal probability, implying higher flexibility and


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Biochemistry

conformational entropy. The MD simulations thus indicate that the V67L mutation reduces active-site dynamics, which may lead to enhanced activity. A

2IN0 ∆∆Ihh-L67

120

120

100

100

Residue Index

Residue Index

2IN9 ∆∆Ihh-V67

80

60

80

60

40

40

20

20

20

40

60

80

100

20

120

40

Residue Index

60

80

100

120

Residue Index 0.000

Contact Probability

1.000

B 0.06

0.06

V67

Run 1 Run 2

0.05 0.04

Run 3

0.03

0.02

0.02

0.01

0.01

-150

-100

-50

0

50

100

150

0.05

0

C1

0.03

0.02

0.02

0.01

0.01

-150

-100

-50

0

50

100

150

-50

0

50

100

150

C1

0

-150

-100

-50

0

50

100

150

0.06

0.06

D121

0.05

D121

0.05 0.04

0.04 0.03

0.03

0.02

0.02

0.01

0.01 -150

-100

-50

0

50

100

150

0.05

0

-150

-100

-50

0

50

100

150

0.05

N139

0.04 0.03

0.03 0.02

0.01

0.01

-150

-100

-50

0

50

100

150

N139

0.04

0.02

0

-100

0.04

0.03

0

-150

0.05

0.04

0

L67

0.05 0.04

0.03

0

Population

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0

-150

-100

-50

0

50

100

150

χ1 Dihedral Angle

Figure 4. MD simulations show that ∆∆Ihh-V67 active site residues experience more conformational dynamics in the side chain, as compared to ∆∆Ihh-L67. (a) Contact maps of ∆∆Ihh-V67 (left) and ∆∆Ihh-L67 (right) from MD simulation, respectively. Warm colors indicated high contact probability; cold colors indicate low contact probability. (b) χ1 dihedral angle distribution for 4 residues V/L67, C1, D121, and N139. The last three are active


16

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Biochemistry

site residues of intein. Each MD simulation was run 3 times, with black, red, and green curves representing the χ1 distribution of each run.

Protein stability has a complicated relationship with protein activity and in many cases, they are uncoupled40, as in c-Myb R2 mentioned above. In the inteins studied here, however, the V67L mutation not only stabilizes the intein fold but also increases intein activity. An attractive explanation is cavity-mediated allosteric effects. In NMR chemical shift perturbation experiments, V67L affects sites far from the site of mutation, including the intein active site14. This was previously termed a “ripple effect”12 and likely contributes to the activation mechanism of V67L. However, it is not clear what the structural pathway of the allosteric effect is. As cavities connect remote sites in a protein, perturbation at one side of the cavity can be relayed to the other side by changes in cavity size, shape, and dynamics. Indeed, residues lining the same cavity were shown to be highly correlated to each other in a xenon trapping experiment of formylglycinamide synthetase41. In the Mtu RecA intein, the active site is composed of four residues: C1, H73, D121 and N13930,42. As shown in Fig. 5, H73 and L67 border the same cavity. C1 and L2 bridge the L67 cavity and another cavity, which is lined by D121 and N139. Thus the V67L mutation may rearrange active site geometry and dynamics to enhance intein activity through its effects on cavities. Couplings between cavities in proteins have indeed been demonstrated by molecular dynamics43. Consistent with these ideas, our previous work showed that L2 and N139 experience some of the largest chemical shift perturbations caused by V67L, away from the site of mutation14. An additional possibility is that V67L may affect the inteinextein junctions and intein-extein interaction, which may shift active site conformation and dynamics to favor catalysis.


17

Biochemistry

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H73 L67

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N139 C1

D121

L2

Figure 5. Activating pathways of ∆∆Ihh-L67 mediated by cavities, shown as orange, semi-transparent surfaces. The intein active site is composed of C1, H73, D121 and N139 (shown in magenta). Both L67 (shown in blue, behind a cavity) and H73 border the same cavity, while C1 and L2 bridge the L67 cavity and another cavity, which is lined by D121 and N139.

In summary, using GdnHCl denaturation, high-pressure NMR, and MD simulations, we demonstrated that the V67L mutation decreases the volume change of folding by 13 ± 3 mL mol1

, roughly the size of a methylene group, validating the cavity-filling hypothesis of V67L

stabilization. In addition, cavity changes may contribute to the activation mechanism of V67L, through allosteric reduction of side chain dynamics at the active site, shown by MD simulation. As inteins have broad applications in biotechnology, more stable inteins can be useful. Our results suggest filling cavities could be a design principle for engineering more stable inteins.


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Biochemistry

AUTHOR INFORMATION Corresponding Author Chunyu Wang. Address correspondence to [email protected]. Author Contributions C.W., A.S.Z. designed the experiments; A.S.Z., Z.L. and M.F. carried out the experiments; D.J.R. and A.G. performed and analyzed MD simulation experiments; A.S.Z. and O.A. analyzed the fluorescence and NMR data; A.S.Z., C.W., K.V.M. and C.A.R wrote the paper. Funding Sources This work was supported by the National Science Foundation (grants MCB-1244089 and MCB1517138 to KVM). ABBREVIATIONS Mtu, Mycobacterium tuberculosis; NMR, Nuclear Magnetic Resonance; HSQC, heteronuclear single quantum coherence; CBD, chitin-binding domain; Cm, center of mass; GdnHCl, guanidine hydrochloride; SDS, sodium dodecyl sulfate; IPTG, isopropyl β-D-1-thiogalactopyranoside; DTT, dithiothreitol; MD, molecular dynamics.


19

Biochemistry

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xenon trapping and statistical coupling analysis. PLoS One 8. (42) Du, Z., Zheng, Y., Patterson, M., Liu, Y., and Wang, C. (2011) pKa coupling at the intein active site: implications for the coordination mechanism of protein splicing with a conserved aspartate. J. Am. Chem. Soc. 133, 10275–10282. (43) Barbany, M., Meyer, T., Hospital, A., Faustino, I., D’Abramo, M., Morata, J., Orozco, M., and De La Cruz, X. (2015) Molecular dynamics study of naturally existing cavity couplings in proteins. PLoS One 10.


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V67L Mutation Fills an Internal Cavity To Stabilize RecA - American

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