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Combining H/D exchange mass spectrometry and computational docking to derive the structure of protein-protein complexes Victoria A. Roberts, Michael E Pique, Simon Hsu, and Sheng Li Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00643 • Publication Date (Web): 03 Nov 2017 Downloaded from http://pubs.acs.org on November 16, 2017

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Biochemistry

Combining H/D exchange mass spectrometry and computational docking to derive the structure of protein-protein complexes

Victoria A. Roberts,

,

Michael E. Pique, Simon Hsu,

,

and Sheng Li

San Diego Supercomputer Center, University of California, San Diego, La Jolla, California

Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, California

School of Medicine, University of California, San Diego, La Jolla, California Current address: Department of Internal Medicine, University of California, San Diego, La Jolla, California E-mail: [email protected] Phone: 858 784-8028

Running header Combining H/D exchange mass spectrometry and computational docking to derive the structure of protein-protein complexes

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Biochemistry

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Abstract Protein-protein interactions are essential for biological function, but structures of proteinprotein complexes are difficult to obtain experimentally. To derive the protein complex of the DNA-repair enzyme human uracil-DNA-glycosylase (hUNG) with its protein inhibitor (UGI), we combined rigid-body computational docking with hydrogen/deuterium exchange mass spectrometry (DXMS). Computational docking of the unbound protein structures provides a list of possible three-dimensional models of the complex; DXMS identifies solventprotected protein residues. DXMS showed that unbound hUNG is compactly folded, but unbound UGI is loosely packed. Increased solvent protection of hUNG in the complex was localized to four regions on the same face. The decrease in incorporated deuterons was quantitatively interpreted as the minimum number of main-chain hUNG amides buried in the proteinprotein interface. Deuteration of complexed UGI decreased throughout the protein chain, indicating both tighter packing and direct solvent protection by hUNG. Three UGI regions showing the greatest decrease were best interpreted leniently, requiring just one main-chain amide from each in the interface. Applying the DXMS constraints as filters to a list of docked complexes gave the correct complex as the largest favorable-energy cluster. Thus, identification of approximate protein interfaces was sufficient to distinguish the protein complex. Surprisingly, incorporating the DXMS data as added favorable potentials in the docking calculation was less effective at finding the correct complex. The filtering method has greater flexibility, with the capability to test each constraint and enforce simultaneous contact by multiple regions, but with the caveat that the list from the unbiased docking must include correct complexes.

INTRODUCTION Improvements in X-ray crystallography and NMR spectroscopy have led to a rapid increase in the determination of individual protein structures. Protein complexes, however, remain difficult tar2


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gets, driving the need for methods that can derive protein-protein complexes based on the structures of unbound proteins. Significant progress in predicting protein-protein complexes has been driven, in part, by the CAPRI docking challenge, 1–3 but computational methods alone cannot yet reliably predict a specific interaction. Incorporating experimental data greatly enhances the likelihood of a good prediction. We explored two approaches for combining data from hydrogen/deuterium exchange mass spectrometry (DXMS) with rigid-body macromolecular docking of the unbound protein structures to develop a structural model of the protein complex. DXMS has proved a powerful method for studying protein interactions. DXMS takes advantage of the huge variation in rates of backbone amide hydrogen exchange with solvent, from a half-life of about one second for an unstructured polypeptide to rates up to 10 slower in structured regions of a protein. Amides in unstructured regions of a protein or having direct interactions with solvent will exchange more readily than those involved in hydrogen bonds, buried within the protein interior, or protected in intermolecular interfaces. In DXMS, hydrogen/deuterium exchange is followed by protein proteolysis. The resulting peptides are analyzed by mass spectrometry, revealing the degree of solvent exchange of backbone amide hydrogen atoms throughout the protein chain. By examining a protein in free and bound states, binding surfaces can be identified. One advantage of DXMS is that proteins are examined under solution conditions similar to those used for activity and binding studies. Further, only small samples ( g) are needed and there is no limitation on protein size. 4 We examined the well-studied interaction of the essential DNA-repair enzyme uracil-DNAglycosylase (UNG) with the uracil-DNA-glycosylase inhibitor protein (UGI). UNG detects uracil in DNA and hydrolyses the N-glycosylic bond between uracil and the deoxyribose, the first step of DNA repair. UGI is an acidic protein of 84 amino acids from the Bacillus subtilis bacteriophage that inactivates UNG from diverse organisms, including B. subtilis, E. coli, and humans. 5 Studies

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on UGI binding to UNG from E. coli show formation of an essentially irreversible complex that requires protein denaturation to separate. 6,7 High-resolution crystallographic structures of human UNG (hUNG), alone 8 and bound to UGI, 9 and of E. coli UNG (ecUNG), alone 10 and bound to UGI, 11,12 show little change in UNG upon UGI binding. The crystallographic structures of free UGI 11 and UNG-bound UGI, however, reveal significant structural differences on the UGI face contacting UNG, making prediction of the UNG-UGI complex computationally challenging. Our goal was to develop methods that effectively combine DXMS data with computational docking of the unbound protein structures. The DXMS data was both applied as a filter to an already computed list of complexes and incorporated directly into the docking calculation as a set of potentials. Applying the experimental data as a set of filters was better at identifying the correct complex, but this approach requires that the unbiased computed list must include some correct complexes.

MATERIALS AND METHODS Proteins preparation for deuterium exchange mass spectrometry (DXMS) UGI and the full catalytic domain of hUNG were expressed and purified as previously described. 13,14

In the hUNG construct, 85 N-terminal residues were replaced by a 22-amino-acid His tag

(MGSSHHHHHHSSGLVPRGSHMG). The final hUNG stock solution had a protein concentration of 9.9 mg/ml (0.36 mM) in a buffer of 10mM TRIS, 10mM NaCl, 1mM DTT, pH 7.5. The final UGI stock solution had a protein concentration of 7.6 mg/ml (0.80 mM) in the same buffer.

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Establishing optimal proteolysis conditions for DXMS We first determined the concentrations of the denaturant guanidine hydrochloride that would, upon proteolysis, give overlapping peptides that spanned the full sequences of hUNG and UGI. Optimally, peptides should be long enough for unique identification but short enough to localize changes in solvent protection. The procedure has been previously described for hUNG. 15 Briefly, a 5 l sample of the protein stock solution was diluted with 15 l of 8.3 mM Tris, 50 mM NaCl (pH 7.2), and then mixed with 30 l of quench solution (0.08 M, 0.8 M, 1.6 M, 3.2 M, or 6.4 M guanidine hydrochloride in 0.8% (v/v) formic acid, 16.6% (v/v) glycerol) on ice. The quench solution brings the samples to pH 2.5 and quenches the deuterium exchange. 16 After quenching, samples were transferred to dry ice within one minute and stored at -80 C until they were transferred to

the dry ice-containing sample basin of the cryogenic autosampler module of the DXMS apparatus. Samples were individually melted at 0 C, then injected (45 l) and pumped through a protease

column (0.05% (w/v) trifluoracetic acid (TFA) at 100 l/min, with 40 s exposure to protease) containing immobilized porcine pepsin (coupled to 20AL support from PerSeptive Biosystems at 30 mg/ml; 66 l column bed volume). Protease-generated fragments were collected on a C18 HPLC column, and eluted by a linear acetonitrile gradient (8%-48% (v/v)), using solvent A (0.05% TFA) and solvent B (80% (v/v) acetonitrile, 20% water, 0.01% TFA). Samples were then injected directly into an LCQ Classic (Thermo Finnigan Inc.) electrospray ion trap-type mass spectrometer. The SEQUEST program (Thermo Finnigan Inc.) was used to identify the likely sequence of the parent peptide ions and these tentative identifications were tested with specialized DXMS data reduction software developed in collaboration with Sierra Analytics, LLC, Modesto, CA. Fragmentation maps for all concentrations of denaturant were generated. The best results were obtained using 0.8 M guanidine hydrochloride (0.5 M final concentration) in the quench solution.

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Deuterium exchange mass spectrometry (DXMS) After establishing good peptide fragmentation, hUNG and UGI were subjected to hydrogen/deuterium exchange experiments followed by mass spectrometry analysis to determine the degree of deuteration of backbone amide hydrogen atoms. To analyze UGI-bound hUNG, hUNG was mixed with UGI in ratio of 2:1, and to analyze hUNG-bound UGI, UGI was mixed with hUNG in a ratio of 2:1. Both mixtures were incubated at room temperature for 90 min, then cooled to 0 C. The in

dividual proteins and the two complex mixtures were then prepared and processed as described above, except that 5 l of each protein solution was diluted with 15 l of deuterium oxide (D O)

buffer containing 1.7 mM Tris, 10 mM NaCl, pD (read) 7.1. Samples were incubated for 10, 30, 100, 300, 1000, 3000, and 10,000 s at 0 C and for 1,000, 3,000, 10,000, and 30,000 at room

temperature. Hydrogen exchange rates are about 10 times faster at room temperature, 17 so these experiments are equivalent to 10,000, 30,000, 100,000, and 300,000 s at 0 C. Data are reported in

terms of the deuteration times at at 0 C. The exponential distribution of deuteration times allows us

to sample both fast and slow exchanging amide protons. Samples were quenched with 30 l of 0.5 M (final concentration) guanidine hydrochloride in 0.8% (v/v) formic acid, 16.6% (v/v) glycerol, then proteolyzed, separated, and analyzed by mass spectrometry. Data for deuterated sample sets were collected in MS1 profile mode in a single, automated 8 hour run, and peptide identification data were collected in data-dependent MS2 mode. 18 Non-deuterated and fully deuterated samples were processed and analyzed for comparison. For the non-deuterated samples, 5 l of each protein solution was mixed with 15 l of 1.7 mM Tris

(pH 7.1), 10 mM NaCl on ice. For the fully deuterated samples, 5 l of each protein solution was mixed with 15 l 0.5% formic acid in D O for 24 hours at room temperature. These conditions al

low complete exchange of backbone amide protons for deuterium, providing, for each peptide, the maximal experimental mass for the fully exchanged peptide under the experimental conditions, in-

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cluding the back exchange that occurs during protein proteolysis and peptide separation by reverse phase chromatography. 19 The mass spectra provide the isotopic profiles of the peptide fragments. Isotopic profiles of non-deuterated peptides have multiple peaks due to the naturally occurring isotopes of C, N, O, and S. Isotopic profiles of deuterated peptides are a combination of the naturally occurring isotopes and the added deuterons, where each deuteron shifts the natural isotropic profile by one unit of mass/charge. Isotopic profiles of three hUNG peptides are shown in Figures S1, S2, and S3, Supporting Information. The centroids of the isotopic envelopes were determined using DXMS data reduction software (Sierra Analytics, LLC, Modesto, CA). The percent deuterium incorporation was calculated as %deuteration incorporation = [m(P) - m(N)] / [m(F)-m(N)] where m(P), m(N), and m(F) are the centroid values of the partially deuterated, non-deuterated, and fully deuterated peptide. The number of deuterons is # of deuterons = %deuterium incorporation

MaxD

where MaxD is the total number of main-chain amides that can retain deuterons: MaxD = (# of amino acids in the peptide) - (# of Pro residues) - 2 The first two amino acids of the peptide do not retain deuterons 16 and the count of proline residues starts at the third residue in the peptide. To check for consistency and correct peptide identification, we examined all overlapping peptides within each data set. Many peptides were present in multiple charge states, each identified and analyzed independently, providing a further check on consistency.

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The DOT calculation Rigid-body docking calculations were performed with the program DOT, 20–22 part of the DOT2 Suite that is distributed by the Computational Center for Macromolecular Structure at the San Diego Supercomputer Center (URL: http://www.sdsc.edu/CCMS). In DOT, one molecule (the moving molecule), represented by its atomic positions with partial atomic charges, is systematically moved within the shape and electrostatic potentials of a second molecule or complex (the stationary molecule), providing an exhaustive translational and rotational search. Interaction energies for all configurations of the two molecules are evaluated as correlation functions, which are efficiently computed with Fast Fourier Transforms. UNG, the larger protein, was assigned as the stationary molecule and UGI was assigned as the moving molecule for computational efficiency. 22 Coordinates for the hUNG/UGI complex (PDB code 1UGH 9 ), unbound hUNG (PDB code 1AKZ), 8 unbound ecUNG (PDB code 1EUG), 10

and unbound UGI (PDB codes 1UGI and 2UGI) 11 were obtained from the Protein Data Bank.

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Molecular properties for UDG and UGI were calculated using utilities in the DOT2 Suite, and

the program REDUCE 24 to add hydrogen atoms, determine His side chain protonation states, and correct the geometry of Asn, Gln, and His side chains; the program MSMS 25 to calculate molecular surfaces that encompass the volumes defining the shape potential of the stationary molecule; the AMBER library of heavy atoms with added polar hydrogens 26 to assign partial atomic charges; and the program UHBD 27 to calculate the electrostatic potential of the stationary molecule by finite difference methods to solve the linearized Poisson-Boltzmann equation. To assign partial charges, the charge states of all residues must be determined. All Asp and Glu side chains were assumed to be negatively charged and all Lys and Arg side chains were assumed to be positively charged. REDUCE assigned the 13 His residues of hUNG as neutral, although the pattern of protonation (ND1 or NE2) differed in the unbound and bound structures. For ecUNG,

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13 His residues were neutral and one (His 136) was positively charged. The single His in UGI was assigned as neutral, but the site of protonation varied among the structures. In the human hUNG construct used in the crystallographic structures, the N-terminal 85 residues have been replaced by the sequence Met-Glu-Phe. Therefore, the N-terminal Met residue was assigned a positively charged N-terminus, as in the construct. The first one or two residues of the UGI structures and the first few residues of ecUNG are not seen in the electron density; therefore a single proton was added to the N-terminal residue to give a neutral N-terminus. The final total charges were +5.0e for each hUNG structure, -2.0e for ecUNG, and -13.0e for each UGI structure. In the electrostatic potential calculations, a dielectric of 3 for the UNG interior, a dielectric ˚ and an ionic strength of of 80 for the surrounding environment, an ion exclusion radius of 1.4 A, 150 mM were used. The electrostatic potential was clamped 28 to make the electrostatic energy compatible with the soft shape fit, resulting in a range of -2.1 to 1.9 kcal/mol/e for bound hUNG, -2.2 to 2.0 kcal/mol/e for unbound hUNG, and -2.2 to 2.2 for ecUNG. The small variations in the clamping values did not affect docking results. ˚ on a side with 1 A ˚ grid spacing (about 2.1 million Docking calculations used a cubic grid 128 A points). UGI was centered at each grid point in 54,000 distinct orientations (a 6.0 rotational

spacing), giving about 108 billion placements of UGI about UNG. The interaction energy was calculated for each placement as the sum of the electrostatic and van der Waals intermolecular energy terms. The electrostatic energy was calculated as the set of point charges at the atomic coordinates of heavy and polar hydrogen atoms of UGI placed in the electrostatic potential of UNG. 20

The van der Waals term was determined by the number of UGI heavy atoms that overlapped a

˚ thick favorable layer around the excluded volume of UNG, which is defined as all grid points 3A inside the UNG molecular surface. Each UGI atom within the favorable layer contributes -0.1 kcal/mol to the van der Waals energy. 22 For dockings that used coordinates from the complex,

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no UGI atoms were allowed to penetrate the UNG excluded volume (0 collisions allowed). For dockings that used unbound coordinates, up to ten UGI atoms were allowed to penetrate the UNG excluded volume (10 collisions allowed). This more lenient shape fit allows for the conformational differences between bound and unbound structures.

Analysis of Docking Results For each placement of the moving molecule, DOT outputs the intermolecular energy and the grid point coordinates and 3 rotations (Euler angles) that, when applied to the starting reference position, generate the UGI position relative to the stationary, reference UNG position. This list, the “E6D” file, provides a compact way to store and analyze a large number of configurations. For the UNG-UGI runs, the 2,000 top-ranked placements were selected for detailed analysis. To analyze the quality of the fit, root-mean-square deviations (RMSDs) of the C atoms of

UGI were calculated. When UGI coordinates were taken from the crystallographic structure of the hUNG-UGI complex (1UGH), the RMSD was calculated between docked and crystallographic ˚ or less considered correct. For unbound UGI coordinates, positions of UGI, with an RMSD of 3 A the RMSD was calculated between the docked UGI molecule and the best fit of the same UGI ˚ or less considered correct. The molecule to the position of UGI in 1UGH, with an RMSD of 4 A RMSD value was incorporated into the E6D file, so that the quality of the fit was linked to each configuration, facilitating further analyses.

Filtering lists of docked complexes with DXMS data DXMS gives the number of main-chain N atoms within a peptide that become more protected in the complex, but cannot distinguish the specific residues. We developed the DOT2 utility dotxyzfilter, 22

which allows detailed queries to be made such as “find all configurations in which at least 4 10


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˚ of any atom of UGI.” Both the main-chain N atoms of hUNG residues 142-158 are within 7 A input and the output of DOTxyzfilter are E6D files, so that individual experimental constraints can ˚ or less between specified backbone be analyzed and easily combined. A maximum distance of 7 A N atoms of one molecule and any nonhydrogen atom of the other molecule gave the best balance between retaining correct configurations and removing false positives. A maximum distance of 6 ˚ removed many correct configurations whereas a maximum distance of 8 A ˚ retained more false A positives. The number of buried main-chain amides found by DXMS for hUNG peptides 142-158, 160-170, 210-220, and 265-274 were used to screen the docking of hUNG with 1UGI, molecule D (Table 2). Proline residues were excluded from the count because they have no exchangeable amide proton. This was particularly important for residues 160-170, in which residues 163 and 165-168 are Pro. Therefore the one additional main-chain N atom that is protected in the complex must come from just six residues: 160-162, 164, 169, or 170. Each hUNG peptide was evaluated with a range of required main-chain atoms to determine the effectiveness of using the number of main-chain amides indicated by DXMS (Table S1, Supporting Information). Peptide 258-274 was included in this analysis to verify that residues 265-274 were solely responsible for the for the increased protection in the complex, as indicated by our analysis of overlapping peptides. Peptides 258-274 and 265-274 gave almost identical numbers of hits, but peptide 265-274 eliminated more incorrect configurations (Table S1, Supporting Information). Extending the hUNG DXMS data to ecUNG required identifying the corresponding four peptides: residues 61-77, 79-89, 129-139, and 184-193. The main-chain N atoms allowed in the count from each peptide corresponded to the non-Pro residues in the hUNG sequence.

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Incorporating DXMS results as potentials in the DOT calculation The DXMS data for UNG was represented as spheres filled with favorable potential surrounding the main-chain N atoms of hUNG peptides 142-158, 160-170, 210-220, and 265-274 and ecUNG peptides 61-77, 79-89, 129-139, and 184-193. No spheres were added for N atoms that corresponded to Pro in hUNG. Grid points within these spheres were added to the list of grid points in ˚ the 3 A-thick favorable layer surrounding UNG, which have a value of 1.0. Grid points within the spheres that lay in the UNG excluded volume remained part of the excluded volume. For hUNG, ˚ and values of 1.0, 1.5, and 2.0 were examined, For example, given a sphere radii of 6, 7, and 8 A sphere with a value of 2.0, each atom from a docked UGI lying within this sphere would contribute ˚ or less or a value greater than 2.0 -0.2 kcal/mol to the van der Waals energy. Using radii of 6 A decreased the number of highly ranked correct configurations compared with the unbiased DOT run. The two sets of radii and values that worked best for hUNG were applied to the docking of ˚ with a value of 1.5 and a radius of 8 A ˚ with a ecUNG with 1UGI, molecule D: a radius of 7 A value of 1.0.

RESULTS DXMS and computational docking were applied to UGI and the catalytic domain of UNG. Four DXMS experiments were done. UGI and hUNG were each examined in the unbound state. Then two experiments were performed on the complex: hUNG was analyzed in the presence of excess UGI, and UGI was analyzed in the presence of excess hUNG. These experiments ensured that the protein under analysis was in the fully bound state. Comparison of the unbound and bound states of each protein revealed regions that undergo a change in solvent exposure in the complex. Rigid-body dockings were performed on coordinates from the hUNG/UGI complex and from the

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unbound structures of hUNG, closely related ecUNG (58% sequence identity), and UGI. In the dockings, UGI (the moving molecule) was translated and rotated around UNG (the stationary molecule) in a systematic search with the program DOT. 20–22 The intermolecular energies for over 100 billion positions of UGI were calculated, and the the 2000 best-energy UGI positions were retained, ranked by their energies. The DXMS data was applied in two ways to the least successful dockings of unbound UGI with unbound hUNG and ecUNG. First, the DXMS data on both hUNG and UGI were interpreted as distance constraints that were then applied as filters to the list of computed complexes. Second, the DXMS data on hUNG was directly incorporated into the DOT docking calculation as added favorable potentials.

DXMS on hUNG and UGI UGI and hUNG alone and the two pre-formed hUNG/UGI complexes were examined by DXMS. In all four experiments, the protein(s) were exposed to deuterium oxide (D O) for 10 time points ranging from 10 s to 300,000 s. The samples were then quenched and subjected to protease digestion. The resulting peptides were separated by HPLC and analyzed by electrospray mass spectrometry. Unbound hUNG gave 128 peptides and bound hUNG gave 110 peptides. The two data sets shared 78 peptides, allowing direct comparison of the change in deuteration. Unbound UGI gave 99 peptides, bound UGI gave 77, with 61 present in both data sets. The percentage deuteration for each peptide was determined by comparison with fully deuterated samples. Since the first two amide protons of each peptide exchange rapidly under the experimental conditions that follow the deuteration step, 16,29 neither contributes to the deuteration count. For example, the mass envelope corresponding to hUNG peptide 140-158 provides deuteration information only for residues 142-158. The data is reported using the peptide range for which there is deuteration information (peptide 142-158), rather than the full peptide.

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B

C Figure 1: Percent deuteration incorporation for hUNG peptides after 30 s (black), 300 s (green), and 10,000 s (magenta) are shown for (A) hUNG alone and (B) UGI-bound hUNG. (C) The change in deuteration between hUNG and UGI-bound hUNG is shown for peptides common to both data sets. A negative percentage indicates less deuteration (increased solvent protection) in the UGIbound hUNG complex. The largest decreases in deuteration occur in hUNG residues 142-158, 160-170, 210-220, and 258-274. Centroids for hUNG peptides at all deuteration times are given in Tables S2, S3, and S4, Supporting Information.

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A

B

C Figure 2: Percent deuteration incorporation for UGI peptides after 30 s (black), 300 s (green), and 10,000 s (magenta) are shown for (A) UGI alone and (B) hUNG-bound UGI. (C) The change in deuteration between UGI and hUNG-bound UGI. At long deuteration times, the largest decreases occur in UGI residues 18-23, 43-47, and 60-70. Centroids for UGI peptides at all deuteration times are given in Tables S5, S6, and S7, Supporting Information.

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DXMS revealed that the individual proteins have distinctly different behaviors in solution. The behavior of unbound hUNG is consistent with a folded structure having a well-defined interior. After deuteration for just 30 s, hUNG showed significant protection ( 30% deuteration), except

for the N-terminus and the large surface loop formed by residues 245-274. (Figure 1A and Figure S4, Supporting Information). After deuteration for 10,000 s, some peptides still retained strong protection (

20% deuteration), while others showed up to 90% deuteration, indicating a wide

range of environments. In contrast, the entire chain of unbound UGI showed a similar degree of solvent exposure for each deuteration time (Figure 2A and Figure S5, Supporting Information). Peptide 56-57, which is in the center of the protein on the middle -strand of the -sheet, was

only somewhat more protected than adjacent, solvent-exposed peptide 60-70. After 300 s, no UGI peptides had less than 20% deuteration, and after 10000 s, no regions had less than 60% deuteration. The significant solvent protection after 30 s deuteration (generally 10-30%) indicates some structure, but the lack of strong protection after 10,000 s of deuteration (

65% for all

peptides) shows an absence of a well-defined interior. In the complex, hUNG showed significant decreases in deuteration in four adjacent peptides: residues 142-158, 160-170, 210-220, and 245-274 (Figure 3). These peptides include all of the hUNG residues that contact UGI in the crystallographic structure of the complex, 9 except residues 275 and 276, for which there was no DXMS data. Residues 142-158 (purple) showed a decrease of at least four deuterons for deuteration times from 300 to 30,000 s. Residues 160-170 (green) showed a decrease of one deuteron for deuteration times less than 10,000 s, and a decrease of two deuterons at longer times. Residues 210-220 (magenta) showed a decrease of three to four deuterons for deuteration times of 300 s or greater. Analysis of small, overlapping peptides revealed that peptide 258-274 (orange), which had a decrease of at least 5 deuterons at deuteration times of 300 s or greater, fully explained the decreased deuteration observed for residues 245-274.

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A

B

Figure 3: Distribution of hUNG regions showing significant solvent protection in the presence of UGI. (A) Residues with significant solvent protection in the presence of UGI mapped onto the C trace (gray) of hUNG: residues 142-158 (purple), 160-170 (green), 210-220 (magenta), and 258-274 (orange). All lie on the face of hUNG that binds UGI (yellow, thin tubes showing the C trace). (B) Deuteration profiles for the same residue ranges, colored as in A.

Since peptide 251-264 showed no change in the complex, the reduced deuteration could be further localized to residues 265-274. The rest of hUNG showed little change in deuteration, consistent with protection of a single face of hUNG by bound UGI. In the complex, UGI showed a dramatic increase in solvent protection throughout the protein chain (Figure 2B and Figure S5, Supporting Information). At 10,000 s, bound UGI showed a wide range of deuteration (10% to 85%), indicating much greater structural stability than unbound UGI. The largest decreases in deuteration occurred in five regions: peptides 18-23, 45-53, 56-57, 60-70, and 82-84. At a deuteration time of 300 s, residues 18-23 (Figure 4A, blue) and 60-70 (orange) both showed a decrease of about 3 deuterons (Figure 4B). The decrease seen for peptide 45-53 could be localized to overlapping peptide 43-47 (green), which showed a decrease of about 2 deuterons at a deuteration time of 300 s (Figure 4B). In crystallographic structures, peptides 18-23, 43-47, and 60-70 lie on one face of UGI (Figure 4A), with residues 18-23 forming edge strand 1, residues

43-47 lying on adjacent strand 2, and residues 60-70 forming a loop that extends over 1 and

17


Biochemistry

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A

Page 18 of 43

B

C Figure 4: Distribution of UGI regions showing significant solvent protection in the presence of hUNG. (A) Residues with significant solvent protection in the presence of hUNG mapped onto the C trace (yellow) of UGI: residues 18-23 (blue), 43-47 (green), 56-57 (pink), 60-70 (orange), and 82-84 (red). Residues 18-23, 43-47, and 60-70 include direct contacts with hUNG (gray tubes, right). Residues 56-57 are in the protein interior and 82-84 lie on the face opposite the hUNGbinding site. (B) Deuteration profiles for residues 18-23, 43-47, and 60-70, colored as in A show decreases of 2 to 4 deuterons in the presence of hUNG for deuteration times of 300 s and longer. (C) Deuteration profiles for the small peptides representing residues 56-57 and 82-84 show a decrease of about 1 deuteron for deuteration times of 1000 s and longer.

18


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Biochemistry

2. Because of the contacts among these three regions, they were selected as the most likely site

for UNG binding. The increased solvent protection of peptides 56-57 and 82-84 corresponded to a decrease of one deuteron for each of these small peptides (Figure 4C). Residues 56-57 are centered in the UGI interior, and therefore are unlikely to contact hUNG. C-terminal residues 82-84, part of edge strand 5, are on the protein surface but lie on the opposite face from 1.

Computational dockings Table 1: Analysis of UNG-UGI dockings Run

Molecules (PDB ID, chain ID)

State

Collisions

RMSD

allowed

cutoff ˚ 3A

1

hUNG (1UGH): UGI (1UGH)

bound

0

2

hUNG (1AKZ): UGI (1UGH)

unbound/bound

0

3

hUNG (1AKZ): UGI (1UGH)

unbound/bound

10

4

hUNG (1AKZ): UGI (1UGI, A)

unbound

10

hUNG (1AKZ): UGI (1UGI, D)

unbound

10

6

hUNG (1AKZ): UGI (1UGI, E)

unbound

10

7

hUNG (1AKZ): UGI (2UGI, A)

unbound

10

8

hUNG (1AKZ): UGI (2UGI, B)

unbound

10

ecUNG (1EUG): UGI (2UGI, A)

unbound

ecUNG (1EUG): UGI (1UGI, D)

unbound

5 (DXMS)

9 (DXMS)

10 (DXMS)

˚ 4A ˚ 4A

Top 30 Hits 29

Best rank (RMSD) ˚ 1 (1.4 A)

14 26

˚ 4A ˚ 4A

13

˚ 4A ˚ 4A

19

12 18

˚ 1 (2.5 A) ˚ 1 (3.4 A) ˚ 3 (4.0 A) ˚ 1 (3.9 A) ˚ 1 (3.8 A) ˚ 1 (3.2 A)

21

10

˚ 4A ˚ 4A

8

˚ 1 (3.2 A) ˚ 4 (3.6 A)

10

˚ 4A

4

˚ 10 (3.8 A)

The RMSD between docked UGI and UGI in the crystallographic complex was calculated for the UGI C atoms (residues 3 - 84). The number of complexes that satisfied the RMSD cutoff in the 30 top-ranked docked solutions. Runs selected to be filtered by DXMS data (see Table 2).

Our goal was to identify the least successful dockings of the unbound proteins, which would be test cases for the application of the DXMS experimental data. We first docked the bound hUNG/UGI coordinates, which should provide the correct complex because both molecules have undergone the induced fit required for complex formation. Then bound UGI was docked to un19


Biochemistry

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Page 20 of 43

bound hUNG to test DOT’s methodology for accommodating the lack of induced fit: a specified number of moving molecule heavy (non-hydrogen) atoms can be allowed to penetrate the molecular surface of the stationary molecule. For UGI, up to 10 atoms of UGI were allowed to collide with hUNG. For the dockings of both unbound proteins, the 10 distinct structures of unbound UGI were first evaluated. Then a representative of each distinct UGI conformation was docked to unbound hUNG. The unbound two UGI structures that gave the worst results were then docked to unbound ecUNG. The 2,000 top-ranked complexes from each docking run were evaluated. The RMSD between the docked and crystallographic positions of UGI was calculated, keeping UNG ˚ or less. For unbound coorfixed. For bound coordinates, a hit was defined by an RMSD of 3 A ˚ or less. Although an RMSD cutoff of 5 A ˚ for C dinates, a hit was defined by an RMSD of 4 A

atoms has been proposed to define a successful hit for two unbound protein structures, 30 we found ˚ and 5 A ˚ showed incorrect residue-residue that some UGI placements with RMSDs between 4 A ˚ interactions throughout the hUNG/UGI interface. Therefore we selected the more conservative 4 A RMSD cutoff. The docking using coordinates from the hUNG-UGI crystallographic complex clearly identified the bound interaction. Since both proteins have undergone induced fit, no collisions were allowed. Among the 2000 top-ranked complexes from DOT, 146 docked placements of UGI were ˚ of the crystallographic position and 189 were within 4 A. ˚ The crystallographic comwithin 3 A plex was clearly identified, with 29 of the 30 top-ranked UGI placements having RMSD values of ˚ or less (Table 1, run 1). We previously reported 17 hits spread throughout the top 500, with 3A rank 3 being the first hit. 21 The dramatic improvement is due to enhancements in the DOT shape potentials, 22 particularly using the molecular surface, rather than van der Waals spheres, to define the excluded volume of UNG. Docking bound UGI to unbound hUNG (PDB code 1AKZ) 8 also identified the crystallographic

20


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Biochemistry

complex as the largest cluster among the 30 top-ranked configurations (Table 1, runs 2 and 3). ˚ With no allowed collisions (run 2), 14 of the 30 top-ranked UGI placements were within 4 A of the bound UGI position. Allowing up to ten collisions (run 3) compensated for the imperfect shape of unbound hUNG, increasing the size of the correct cluster to 26 of the 30 top-ranked UGI placements (RMSD

˚ The first distant placement (RMSD 4 A).

˚ occurred at rank 58. This 5 A)

is also greatly improved over our initial report, which had just 15 hits in the top 500, with the first hit at rank 13.

A

B

Figure 5: Conformational variation of UGI residue Glu 20 among the 11 crystallographic structures of UGI. A. Glu 20 of the hUNG-bound structure of UGI (yellow) differs in side-chain conformation from one unbound UGI structure (2UGI, molecule A - blue side chain) and in side-chain and main-chain conformation from the other 9 unbound UGI structures (2UGI, molecule B - light blue side chain and C trace; 1UGI, molecule A - orange side chain; 1UGI, molecules B-H - red side chains). When superposed onto bound UGI, the Glu 20 side chains of all 9 unbound UGI structures penetrate the molecular surface of hUNG (green C trace). These create significant steric clashes with side chains extending from one hUNG loop (dark green C loop). B. Glu 20 of unbound UGI (2UGI, molecule B, light blue) demonstrates the poor steric fit to hUNG. When superposed onto bound UGI, the end of the Glu 20 side chain lies in the interior of unbound ˚ of hUNG (green C backbone with gray molecular surface) and the C and C are within 1 A the hUNG surface. Glu 20 of hUNG-bound UGI (yellow) does not penetrate the interior of the unbound hUNG structure. The C backbones of unbound UGI and hUNG were superposed onto the coordinates of the hUNG-UGI complex.

21


Biochemistry

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Page 22 of 43

To select unbound UGI structures for computational docking, we evaluated the conformational variation among the ten distinct UGI structures found in two crystallographic structure files (PDB codes 1UGI and 2UGI). 11 Superposition onto hUNG-bound UGI revealed that the largest structural variation occurred around UGI residue Glu 20 (Figure 5A), which is centered in the hUNG-UGI interface. Molecule A from 2UGI is the closest to the bound UGI structure, with the same backbone conformation, but a different orientation for the Glu 20 side chain (blue, Figure 5A). All other UGI structures have an alternate backbone conformation for residues 18-21, causing the Glu 20 side chain to penetrate into the hUNG interior (Figure 5B). Five UGI structures were selected that spanned the conformational variation of Glu 20: molecule A from 2UGI (blue), molecule A from 1UGI (orange), molecules D and E from 1UGI (red), and molecule B from 2UGI (light blue). Each UGI structure was docked to unbound hUNG, allowing up to 10 collisions. All five dockings identified the crystallographic complex as the largest cluster among the top 30 (Table 1, runs 4 to 8). Molecule D from 1UGI, the unbound UGI structure that most deeply penetrates into the hUNG interior (Figure 5), gave the smallest correct cluster (run 5). Twenty-nine of the top 30 UGI placements docked to the correct face of hUNG (Figure 6A-C). The crystallographic complex was represented by 12 hits (RMSD

˚ Visual analysis found that three additional placements 4 A).

(RMSDs from 4.5 to 6.1) showed a similar orientation of UGI and position for Glu 20, resulting in a cluster of 15 within the top 30 UGI placements. Two UGI molecules (2UGI, molecule A and 1UGI, molecule D) were docked to the structure of unbound ecUNG (PDB code 1EUG). These dockings (Table 1, runs 9 and 10) were less successful than the hUNG runs: but still 20 of the top 30 lay over the correct face of ecUNG. The other 10 were widely dispersed over the ecUNG surface. The correct complex gave the only distinct cluster within the top 30 (Figure 7A).

22


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Biochemistry

Applying DXMS data as a filter to docked UNG-UGI complexes To apply the DXMS data to the list of complexes generated by computational docking, they must be interpreted as distance constraints. In the complex, the four key hUNG regions showed a constant decrease in deuteration over the range from 300 s to 10,000 s: residues 142-158 (four amides), residues 160-170 (one amide), residues 210-220 (three amides), and residues 258-274 (five amides). The observed decrease in deuteration for residues 258-274 was localized to 265274, because peptide 251-264 showed no change in the complex. We interpreted the number of protected amides as the minimum number of main-chain N atoms in each peptide that must be ˚ of any heavy buried in the UNG/UGI interface. A buried atom is defined as being within 7 A atom of UGI. Pro residues do not have exchangeable amide protons, so Pro residues 146, 150, 163, 165-168, 269, and 271 were not included in the count of buried amides. The peptide constraints were applied to the least successful unbound docking of hUNG, which used molecule D from 1UGI (Table 1, run 5). We first evaluated our assumption that the decrease in deuterons at intermediate deuteration times correlates with the number of hUNG amide protons that become buried in the complex. Requiring fewer buried amides (Table S1, Supporting Information) increased the number of retained incorrect configurations, as expected. Increasing the number of buried amides by one gave different results among the peptides. The number of hits in the top 30 and top 100 increased (peptides 142-158 and 160-170) or stayed the same (210-220), but at the cost of eliminating a few hits in the top 2000. For peptide 265-274, or overlapping peptide 258-274, requiring an additional buried amide decreased the number of hits in the top 30 and 100 and eliminated over 60% of the hits in the top 2,000. Since eliminating hits is detrimental, and we cannot tell, a priori, which peptides can tolerate greater constraints, interpreting the DXMS data as the minimum number of amides that must be buried was a reliable strategy for all four peptides. Compared with the initial DOT run (Table 2, filter 1), each hUNG peptide filter increased the

23


Biochemistry

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Page 24 of 43

Table 2: Using DXMS data to filter the 2000 top-ranked UNG-UGI complexes Amide N DXMS filter

Count

Correct in Total

Top 30

Top 100

Total

% Correct

hUNG (1AKZ) with UGI (1UGI, molecule D) 1

DOT, no filter

–

2000

12

25

160

8.1

2

hUNG 142-158

4

1095

12

33

160

14.6

3

hUNG 160-170

1

1242

13

32

160

12.9

4

hUNG 210-220

3

1678

12

27

160

9.5

5

hUNG 265-274

5

1355

14

30

138

10.2

6

All hUNG peptides

4,1,3,5

816

16

37

138

16.9

7

UGI 18-23

1

1817

12

25

160

8.8

8

UGI 18-23

3

1639

12

27

160

9.8

9

UGI 43-47

1

1815

12

25

160

8.8

10

UGI 43-47

2

1756

12

25

160

9.1

11

UGI 60-70

1

1249

12

30

160

12.8

12

UGI 60-70

3

676

11

26

85

12.6

13

All UGI peptides, lenient

1,1,1

1081

12

31

160

14.8

14

All UGI peptides, stringent

3,2,3

579

11

28

85

14.7

15

All hUNG + all UGI (lenient)

588

16

38

138

23.5

16

All hUNG + all UGI (stringent)

408

12

31

72

17.6

–

2000

8

9

77

3.9

4,1,3,5

904

8

13

61

6.8

ecUNG (1EUG) with UGI (2UGI, molecule A) 17

DOT, no filter

18

All ecUNG peptides

19


1,1,1

731

7

15

51

7.0

20


3,2,3

175

0

5

9

5.1

21

All ecUNG + all UGI (lenient)

446

7

16

38

8.5

–

2000

4

7

35

1.8

4,1,3,5

520

7

13

35

6.7

ecUNG (1EUG) with UGI (1UGI, molecule D) 22

DOT, no filter

23

All ecUNG peptides

24


1,1,1

754

7

11

33

4.4

25


3,2,3

323

3

6

11

3.4

26

All ecUNG + all UGI (lenient)

341

7

15

33

9.7

24


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Biochemistry

number of hits among the top 100 (Table 2, filters 2-5). Sequentially applying all four hUNG peptide filters eliminated 1184 of the 2000 complexes, resulting in a list with 17% correct configurations in which the top 30 and top 100 ranked complexes were enriched in correct solutions (Table 2, filter 6). The top 30 are more tightly clustered over the binding site (Figure 6D) than in the original DOT run. The cluster representing the crystallographic complex contained 20 placements: the 16 hits and four additional placements (RMSDs from 4.2 to 6.1). Interpretation of the DXMS data on UGI was more difficult because stabilization contributes to the decreased deuteration observed in the complex. Two filtering approaches were explored, focusing on the three peptides that showed significant decreases in deuteration and form a cluster on one face of UGI: peptides 18-23, 43-47, and 60-70. First, the DXMS data on UGI was interpreted as a stringent filter using the full decrease in deuterons: 3 for peptide 18-23, 2 for peptide 43-47, and 3 for peptide 60-70. This stringent filter assumes that hUNG contacts are the principal cause for increased solvent protection. Second, the DXMS data was interpreted more leniently: just one amide N from each peptide was required to be buried in the interface. The stringent and lenient filters gave similar results for residues 18-23 and 43-47 (Table 2, filters 7-10). However, the stringent filter for residues 60-70 eliminated almost half of the correct configurations (Table 2, filters 11, 12), indicating that structural stabilization of this loop contributes significantly to its increased solvent protection. Combining the three lenient UGI filters (Table 2, filter 13) retained all the correct complexes among the top 2000. Adding the lenient UGI constraints to the the hUNG constraints had little effect on the top 30 and top 100 complexes, but eliminated an additional 228 false positives, resulting in 23.5% correct configurations (Table 2, filter 15). The combined stringent UGI filters alone (Table 2, filter 14) or in combination with the hUNG constraints gave worse results (Table 2, filter 16), eliminating over 50% of the correct complexes. The UGI C-terminus (residues 82-84) is the only other solvent-exposed region in the crystal-

25


Biochemistry

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A

Page 26 of 43

B

C

D

E

Figure 6: The 30 top-ranked placements of unbound UGI (1UGI, molecule D) docked to unbound hUNG. Docked UGI molecules (shown as C trace) are colored by their fit to the UGI in the ˚ light green - 4.0 RMSD 7.0 A, ˚ light crystallographic complex: light blue - RMSD 4.0 A, ˚ Glu 20 side chains are shown in red, except Glu 20 of the highest ranked red - RMSD 7.0 A. UGI placement is blue. (A) The distribution among the 30 top-ranked UGI placements from the unbiased docking. Three placements (left) represent the 29 that docked over the UGI-binding face ˚ a near-by placement (light of hUNG (gray C trace): a hit (light blue, rank 1, RMSD 3.5 A), ˚ ˚ green, rank 11, RMSD 5.8 A), and a more distant placement (light red, rank 3, RMSD 25.1 A). ˚ (B) The UGI interaction One placement (right) was far from the interface (rank 30, RMSD 51.8 A). surface. The view is rotated 90 from (A), and hUNG is not shown. The highest ranked UGI hit (light blue with blue Glu 20) is compared with UGI from the crystallographic complex (yellow) and with unbound UGI (orange, 1UGI, molecule D) fit to UGI in the crystallographic complex. This orientation is shown in (C) - (E) and in Figure 7. (C) The 29 placements that docked to the UGI-binding face of hUNG. Fifteen correctly center Glu 20 in the interface: 12 with RMSD 4 ˚ (all structures with light blue C trace) and three with RMSDs from 4.5 to 6.1. (D) The top A) 30 placements after screening with the hUNG DXMS constraints. All dock to the UGI-binding face of hUNG and the correct cluster contains 20 placements. (E) Top 30 after incorporation of ˚ spheres filled with a favorable value of 1 for the hUNG DXMS into the DOT calculation as 26 8A the hUNG main-chain N atoms. The correct cluster contains 16 placements: 13 hits and three ˚ additional placements (RMSDs from 4.4 to 4.8 A).


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Biochemistry

A

B

C

Figure 7: DXMS data applied to ecUNG/UGI dockings. Docked UGI molecules are colored by their fit, as in Figure 6 and are shown in the same orientation as in Figure 6, (B) - (E). (A) The 20 UGI placements among the top 30 from DOT that bind to the UGI-binding face of hUNG. View is as in Figure 6(B-D), looking at the UGI interaction surface. (B) Top 30 after applying all hUNG constraints to ecUNG. (C) Top 30 with the hUNG DXMS data incorporated into the DOT ˚ spheres calculation in which corresponding ecUNG main-chain N atoms were surrounded by 7 A filled with a favorable value of 1.5. lographic structure that also showed significant protection in the hUNG-UGI complex. Requiring one amide of peptide 82-84 to be in the interface gave 282 complexes out of the top 2000, none close to the bound UGI position. Adding the other three UGI peptides as lenient constraints resulted in only 46 complexes. Adding the hUNG constraints left just 12 complexes, which formed three clusters. These might be considered as possible candidates if the complex structure were unknown. However, it would be reasonable to assume that the change in solvent exposure of residues 82-84, which are part of the C-terminal -strand, is solely due to the increased stabilization of the

-sheet in the complex. We then investigated if filtering could improve identification of the correct complex in the

unbound dockings of UGI with ecUNG, which gave worse docking results than hUNG. The DXMS data on hUNG was applied to the corresponding main-chain N atoms of ecUNG (Table 2, filters 18 and 23). The docking with 1UGI, molecule D, where DOT gave just 4 correct complexes in 27


Biochemistry

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Page 28 of 43

the top 30, was significantly improved, with all 7 correct complexes in the top 100 moving into the top 30. Lenient UGI constraints (Table 2, filters 19 and 24) showed similar improvements, whereas stringent UGI constraints (Table 2, filters 20 and 25) eliminated many correct complexes. Adding the lenient UGI constraints to the hUNG constraints further increased the percentage of correct complexes (Table 2, filters 21 and 26), predominantly by eliminating additional incorrect complexes. As found for hUNG, the correct clusters were larger than indicated by the number of hits. In the DOT run, the cluster corresponding to the crystallographic complex consisted of 4 hits and one ˚ with the other top 30 placements widely dispersed additional UGI placement (RMSD of 4.6 A), (Figure 7A). Applying the four hUNG peptide filters narrowed the distribution of the 30 topranked placements and resulted in two large clusters: one corresponded to the crystallographic ˚ and the complex and contained all 7 hits and two additional placements (RMSDs of 4.5 and 4.6 A) ˚ which was displaced about 6 A ˚ from other containing 11 placements (RMSDs from 6.4 to 9.1 A), the correct cluster (Figure 7B).

DXMS data included as a potential in the DOT calculation Incorporating the DXMS data into the DOT calculation should increase the number of correct, favorably ranked complexes compared with the unbiased DOT run. This approach could be particularly useful for ecUNG, which gave fewer correct complexes in the top 2000 than hUNG. We first explored how best to incorporate the DXMS data on hUNG as a potential. The DOT shape potential of UNG, as the stationary molecule, consists of the excluded volume within the molec˚ ular surface surrounded by a 3 A-thick favorable region, in which grid points have a value of 1.0. The four key hUNG peptides were added to the favorable region of hUNG as spheres filled with a favorable value centered around the backbone nitrogen atoms (except Pro N). We tested the effect

28


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Biochemistry

Table 3: Direct incorporation of hUNG DXMS data into the DOT calculation Amide N spheres Run

Radius

Value

Top 30

6

2.0

7

20

135

6.8

2

7

1.0

12

25

190

9.5

3

7

1.5

11

27

196

9.8

4

7

2.0

11

26

177

8.8

5

8

1.0

13

32

234

11.7

6

8

1.5

11

30

220

11.0

7

8

2.0

10

26

198

9.9

7

1.5

2

6

37

1.9

8

1.0

1

6

37

1.9

1

8

Molecules

Hits in

hUNG: UGI (1UGI, D)

ecUNG: UGI (1UGI, D)

9

Top 100 Top 2000

% Correct

of varying the size of the spheres and the value within them on the docking of UGI (molecule D) ˚ increased the number of correct configurations in the top with hUNG. Sphere radii of 7 and 8 A 2000 compared with the unbiased DOT run (160/2000 hits), with favorable values of 1.0 and 1.5 ˚ spheres filled with a value of 1.0 gave the most hits being the most effective (Table 3). The 8 A in the top 2000 (234, Table 3, run 5), but had fewer hits in the top 30 (Figure 6E) and 100 than the unbiased DOT run followed by screening with the same hUNG peptides (Table 2, filter 6, and Figure 6D). ˚ spheres filled with a value of The sets of values that gave the best results for hUNG (7 A ˚ spheres filled with a value of 1.0) were applied to the corresponding atoms in ecUNG. 1.5, 8 A The unbiased docking of ecUNG with UGI (1UGI, molecule D) gave just 35 hits in the top 2000 (Table 2, run 22). Disappointingly, adding the hUNG DXMS data as potentials gave just 37 hits in the top 2000 and decreased the number of hits in the top 30 and 100. Further, in the dominant ˚ with Glu 20 moved out of the cluster, UGI is in the wrong orientation (RMSDs greater than 20 A), 29


Biochemistry

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interface (Figure 7C). Thus, applying the DXMS data as filters to the unbiased ecUNG docking was a more successful approach, giving the correct complex as one of the two most favorable clusters. We considered the approach suggested by Rey et al., 31 in which residues within a peptide are ranked by their likelihood of being in the interface and the added potentials are weighted accordingly. Unfortunately, examination of the unbound UNG structure showed no clear structural basis for distinguishing among the residues within each UNG peptide. The four key peptides are clustered around the active site; no segments clearly extend away from the active-site pocket. Further, since UGI is an inhibitor, there is no reason to assume that the UGI-binding site is centered over the UNG active-site pocket.

DISCUSSION The combination of hydrogen/deuterium exchange data and computational modeling has proved useful for determining protein-DNA interactions, 15,32 constructing models of amyloid peptide oligomerization, 33–37 and the assembly of pilin proteins into bacterial filaments. 38 DXMS data has been incorporated into computational docking either as added potentials 31 or to restrict the search area for ligand binding. 39 For protein-protein complexes, DXMS has the advantage that it can provide information on the interaction surfaces of both partners. However, DXMS does not provide information on specific interactions between the two proteins, raising the question of whether identification of approximate interaction surfaces is sufficient to distinguish the correct complex. 31 To address this question, we obtained DXMS data on UGI and hUNG and then applied this data to the computational docking of UGI to hUNG and to the closely related bacterial ecUNG. DXMS on unbound hUNG and UGI revealed that these two proteins have distinctly different behaviors in solution. These differences influenced how best to interpret the DXMS data for computational 30


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docking. The solution behavior observed by DXMS for UNG, both unbound and UGI-bound, agrees with crystallographic structures. Even at very long deuteration times, some regions of unbound hUNG showed little deuteration, consistent with a stable tertiary structure and a solvent-protected interior. In the complex, the four hUNG peptides showing increased solvent protection were localized to one face, revealing a potential binding surface for UGI. For each peptide, the decrease in deuterons was interpreted as the minimum number of amides that must be buried in the interface, providing four quantitative filters. Applying these filters to the 2000 top-ranked complexes from the unbiased DOT calculation retained most of the correct complexes, increased the number of correct complexes within the 30 top-ranked configurations, and eliminated many incorrect configurations. Surprisingly, incorporating the DXMS data on hUNG as an added favorable surface in the docking calculation was less effective than filtering an unbiased DOT calculation. The experimentally biased docking increased the number of correct hUNG/UGI complexes in the top 2000, but gave fewer correct complexes in the top 30 and 100 configurations (compare Table 3, runs 3 and 5, with Table 2, filter 6). We expected that ecUNG/UGI system, for which the unbiased docking gave only 35 correct complexes in the top 2,000, would benefit by including the DXMS data in the docking calculation. Instead, the biased docking gave only a slight increase in the total hits (37/2000) and fewer complexes in the top 30 and top 100. Further, the dominant, favorable energy cluster in the biased docking put the wrong face of UGI against ecUNG. In contrast, filtering the unbiased ecUNG/UGI docking with the DXMS data from hUNG increased the number of correct complexes in the top 30 and top 100. The correct complex was one of two closely related clusters that both use the same contact face of UGI and, together, make up 20 of the 30 top-ranked configurations.

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Why was the filtering approach more effective? The experimentally biased docking maximizes the number of UGI contacts with the UNG peptides, but does not take into account a key constraint from the DXMS data: UGI must simultaneously contact the four UNG peptides, with at least a specified number of amides from each peptide in the interface. The filtering procedure incorporates this key constraint, resulting in a more successful outcome. Two inherent properties of the data may influence the biased docking. First, DXMS identifies the regions with increased solvent protection, but cannot distinguish the specific residues responsible for the protection. Parts of the key peptides are outside the contact region, but still are assigned favorable values in the biased docking. Second, DXMS provides no information on proline residues. The latter factor may be particularly important for the hUNG/UGI complex because six hUNG proline residues (165-168, 269, and 271) are near UGI. In particular, hUNG peptide 160-170, with four Pro residues, is underrepresented compared with the other UGI-contacting peptides. In the biased docking, contact with Pro 165-168 becomes less favorable than contact with the favorably weighted regions, moving the best ranked complexes away from this region. In contrast to UNG, the solution behavior of unbound UGI observed by DXMS does not agree with crystallographic structures. In the crystal, both unbound and UDG-bound UGI show the same compact globular fold in which two large loops pack against the central -sheet. We expected

a compact folded structure, and were therefore surprised when initial examination of the DXMS data (Figure S5, Supporting Information) showed a similar degree of solvent exposure throughout the protein chain. This plot, based on a few peptides that span the entire UGI sequence, was validated by plotting the data for all peptides (Figure 2A). The significant solvent protection for most peptides at short deuteration times (30 s) is consistent with retention of secondary structure. The significant solvent exposure throughout the protein at longer deuteration times (10,000 s) shows that these secondary structure elements are not tightly packed. This agrees with the NMR solu-

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tion structure of unbound UGI, 40 which shows defined structures for the central -sheet and two

large loops. However, both loops extend into solvent, exposing the entire -sheet to solvent. In

the crystal, packing and, possibly, the high salt concentration must promote the compact structure of unbound UGI. Our results demonstrate the ability of DXMS to reveal mobility, in this case throughout an entire protein, that is masked in the crystal environment. 41 Further, DXMS explains and resolves the apparent contradiction between crystallographic 11 and NMR 40 structures of unbound UGI, emphasizing the key role that environment has on this small (84-residue), highly charged (-12 e) protein. Unlike the crystallographic structures, both DXMS and NMR 40 show that extensive changes occur throughout UGI upon binding UNG. DXMS provides an interpretation of these changes. Some regions of UNG-bound UGI show significant protection even at long deuteration times, indicating a solvent-protected interior, which is consistent with the compact fold observed for UGI in the crystallographic complex. Thus, in solution, binding to UNG dramatically reduces the flexibility of the UGI tertiary structure. We note that obtaining full sequence coverage in the DXMS experiment was essential for revealing the global structural changes between unbound and bound UGI. Since both overall structural stabilization and direct contacts with UNG contribute to the increased solvent protection of UGI, it was unclear how to best apply the DXMS data to computational docking. Quantitative interpretation of the data ran the risk of overestimating the number of amides that contact hUNG, as seen for UGI peptide 60-70, where the quantitative filter eliminated many correct complexes (Table 2, filter 12). Interpreting the DXMS data on UGI as lenient, qualitative filters proved a better approach, eliminating many incorrect configurations while retaining all correct complexes. Our completed studies on hUNG bound to UGI and to DNA 15 provide an opportunity to com-

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pare the use of DXMS combined with computation docking to protein-protein and protein-DNA complexes. The most effective approaches are different in the two types of systems. In a protein-protein interaction, the unbound structures of each protein, if known, provide good models for the protein structure in the complex. Small differences between the unbound and bound protein structures can make it difficult to identify the correct complex by computational docking alone. The cluster representing the correct complex may be present in the top-ranked configurations, but often must be distinguished from incorrect favorable-energy clusters. Experimental data, such as a known protein-protein contact, can resolve this problem, pulling out the correct cluster. DXMS is particularly valuable because it provides information on both partners. In a protein-DNA interaction, we lack a DNA structure mimicking the bound conformation. However, systematic-search computational docking with small double-stranded DNA fragments (8-11 bp) can reveal the extent of the DNA-binding surface on the protein. Typically, the top several hundred DNA placements found by DOT align in a single cluster that follows the bound DNA position. 28,42 For hUNG, the docked DNA ensemble found by DOT suggested an extensive DNA-binding surface capable of accommodating at least 30-bp of DNA. 15 In contrast, in the crystallographic complex, the bound 11-bp DNA fragment only contacts the hUNG active site. 43 Guided by the computational results, we designed a 30-bp DNA fragment for DXMS. In the resulting hUNG/DNA complex, the active site and two hUNG regions adjacent to the active site showed greatly increased solvent protection, 15 providing definitive proof of an extensive DNA-binding surface on hUNG. This surface extends well beyond the UGI-contact surface. Residues 210-220 are more strongly protected in the hUNG/DNA complex, picking up just one deuteron after a 30,000 s deuteration time. Residues 251-264 become strongly protected in the hUNG/DNA complex, but show the same degree of solvent exposure in unbound and UGI-bound hUNG. DXMS of the complex with the 11-bp DNA fragment used in the crystallographic complex 43

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revealed very different behavior. 42 Solvent exposure of the bound hUNG was similar to unbound hUNG. Increased solvent protection was only observed for peptides containing Leu 272, 42 which is on one lip of the active site and inserts into the DNA base stack. 43 The lack of increased protection in the rest of the active site indicates that the short 11-bp DNA fragment is only loosely held in the active-site. Thus, DXMS again revealed mobility in solution that is not observed in the crystallographic complex, where extensive crystal contacts stabilize an apparent tight complex of the DNA at the hUNG active site. 15 Computational docking was key for defining the extent of the DNA-binding region, providing guidance for designing the 30-bp DNA substrate, which better mimicked the natural, very long DNA substrate.

CONCLUSIONS Combining DXMS with computational docking is a powerful approach for deriving detailed models of protein complexes. DXMS can reveal new structural information, even for extensively studied protein-protein and protein-DNA complexes. DXMS complements x-ray crystallography, providing information on interfaces of complexes that are difficult to crystallize and revealing mobility that may be masked in the crystal environment. Computational docking can assist both the design of the DXMS experiment and the interpretation of the DXMS data in the context of the 3-dimensional structure.

Acknowledgement This paper is dedicated to the memory of Professor Virgil L. Woods, Jr. (1948-2012), who developed the hydrogen/deuterium-exchange mass spectrometry technology used in this work. The authors thank Dr. Lynn Ten Eyck for advice and Dr. Ten Eyck and Dr. Tammy Woo for careful 35


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reading of the manuscript. This work was supported by National Science Foundation grant DBI 99-04559, and National Institutes of Health grants GM070996, GM020501, NS070899, and AI117905. This is manuscript #29543 from The Scripps Research Institute.

Supporting Information Available Effect of varying peptide amide count on the filtering of computed complexes (Table S1), peptide deuteration profiles for hUNG and UGI peptides in the unbound and bound states, and the change in deuteration upon formation of the complex (Tables S2-S7), mass spectra showing isotopic profiles of specific peptides (Figures S1-S3), and deuteration profiles of hUNG and UGI mapped onto the protein sequence (Figures S4 and S5).

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