Propagation of conformational instability in FK506-binding protein FKBP12.

FKBP12 is the archetype of the FK506 binding domains that define the family of FKBP proteins which participate in the regulation of various distinct physiological signaling processes. As the drugs FK506 and rapamycin inhibit many of these FKBP proteins, there is need to develop therapeutics which exhibit selectivity within this family. The long ß4-ß5 loop of the FKBP domain is known to regulate transcriptional activity for the steroid hormone receptors and appears to participate in regulating calcium channel activity for the cardiac and skeletal muscle ryanodine receptors. The ß4-ß5 loop of FKBP12 has been shown to undergo extensive conformational dynamics, and here we report hydrogen exchange measurements for a series of mutational variants in that loop which indicate deviations from a two-state kinetics for those dynamics. In addition to a previously characterized local transition near the tip of this loop, evidence is presented for a second site of conformational dynamics in the stem of this loop. These mutation-dependent hydrogen exchange effects extend beyond the ß4-ß5 loop, primarily by disrupting the hydrogen bond between the Gly 58 amide and the Tyr 80 carbonyl oxygen which links the two halves of the structural rim that surrounds the active site cleft. Mutationally-induced opening of the cleft between Gly 58 and Tyr 80 not only modulates the global stability of the protein, it promotes a conformational transition in the distant ß2-ß3a hairpin that modulates the binding affinity for a FKBP51-selective inhibitor previously designed to exploit a localized conformational transition at the homologous site.

Transient conformations in the unliganded FK506 binding domain of FKBP51 correspond to two distinct inhibitor-bound states.

Members of the FK506-binding protein (FKBP) family regulate a range of important physiological processes. Unfortunately, current therapeutics such as FK506 and rapamycin exhibit only modest selectivity among these functionally distinct proteins. Recent progress in developing selective inhibitors has been reported for FKBP51 and FKBP52, which act as mutual antagonists in the regulation of steroid hormone signaling. Two structurally similar inhibitors yield distinct protein conformations at the binding site. Localized conformational transition in the binding site of the unliganded FK1 domain of FKBP51 is suppressed by a K58T mutation that also suppresses the binding of these inhibitors. Here, it is shown that the changes in amide hydrogen exchange kinetics arising from this K58T substitution are largely localized to this structural region. Accurate determination of the hydroxide-catalyzed exchange rate constants in both the wildtype and K58T variant proteins impose strong constraints upon the pattern of amide exchange reactivities within either a single or a pair of transient conformations that could give rise to the differences between these two sets of measured rate constants. Poisson-Boltzmann continuum dielectric calculations provide moderately accurate predictions of the structure-dependent hydrogen exchange reactivity for solvent-exposed protein backbone amides. Applying such calculations to the local protein conformations observed in the two inhibitor-bound FKBP51 domains demonstrated that the experimentally determined exchange rate constants for the wildtype domain are robustly predicted by a population-weighted sum of the experimental hydrogen exchange reactivity of the K58T variant and the predicted exchange reactivities in model conformations derived from the two inhibitor-bound protein structures.

Molecular Dynamics-Assisted Optimization of Protein NMR Relaxation Analysis.

NMR relaxation analysis of the mobile residues in globular proteins is sensitive to the form of the experimentally fitted internal autocorrelation function, which is used to represent that motion. Different order parameter representations can precisely fit the same set of 15N R1, R2, and heteronuclear NOE measurements while yielding significantly divergent predictions of the underlying autocorrelation functions, indicating the insufficiency of these experimental relaxation data for assessing which order parameter representation provides the most physically realistic predictions. Molecular dynamics simulations offer an unparalleled capability for discriminating among different order parameter representations to assess which representation can most accurately model a wide range of physically realistic autocorrelation functions. Six currently utilized AMBER and CHARMM force fields were applied to calculate autocorrelation functions for the backbone H-N bond vectors of ubiquitin as an operational test set. An optimized time constant-constrained triexponential (TCCT) representation was shown to markedly outperform the widely used (Sf2,τs,S2) extended Lipari-Szabo representation and the more closely related (Sf2,SH2, SN2) Larmor frequency-selective representation. Optimization of the TCCT representation at both 600 and 900 MHz 1H converged to the same parameterization. The higher magnetic field yielded systematically larger deviations in the back-prediction of the autocorrelation functions for the mobile amides, indicating little added benefit from multiple field measurements in analyzing amides that lack slower (∼ms) exchange line-broadening effects. Experimental 15N relaxation data efficiently distinguished among the different force fields with regard to their prediction of ubiquitin backbone conformational dynamics in the ps-ns time frame. While the earlier AMBER 99SB and CHARMM27 force fields underestimate the scale of backbone dynamics, which occur in this time frame, AMBER 14SB provided the most consistent predictions for the well-averaged highly mobile C-terminal residues of ubiquitin.

13C NMR Relaxation Analysis of Protein GB3 for the Assessment of Side Chain Dynamics Predictions by Current AMBER and CHARMM Force Fields.

Molecular simulations with seven current AMBER- and CHARMM-based force fields yield markedly differing internal bond vector autocorrelation function predictions for many of the 223 methine and methylene H-C bonds of the 56-residue protein GB3. To enable quantification of accuracy, 13C R1, R2, and heteronuclear NOE relaxation rates have been determined for the methine and stereochemically assigned methylene Cα and Cß positions. With only three experimental relaxation values for each bond vector, central to this analysis is the accuracy with which MD-derived autocorrelation curves can be represented by a 3-parameter equation which, in turn, maps onto the NMR relaxation values. In contrast to the more widely used extended Lipari-Szabo order parameter representation, 95% of these MD-derived internal autocorrelation curves for GB3 can be fitted to within 1.0% rmsd over the time frame from 30 ps to 4 ns by a biexponential Larmor frequency-selective representation (LF-S2). Applying the LF-S2 representation to the experimental relaxation rates and uncertainties serves to determine the boundary range for the autocorrelation function of each bond vector consistent with the experimental data. Not surprisingly, all seven force fields predict the autocorrelation functions for the more motionally restricted 1Hα-13Cα and 1Hß-13Cß bond vectors with reasonable accuracy. However, for the 1Hß-13Cß bond vectors exhibiting aggregate order parameter S2 values less than 0.85, only 1% of the MD-derived predictions lie with 1 σ of the experimentally determined autocorrelation functions and only 7% within 2 σ. On the other hand, substantial residue type-specific improvements in predictive performance were observed among the recent AMBER force fields. This analysis indicates considerable potential for the use of 13C relaxation measurements in guiding the optimization of the side chain dynamics characteristics of protein molecular simulations.

Pseudomonas infections among hospitalized adults in Latin America: a systematic review and meta-analysis.

BACKGROUND: Treatment of resistant Pseudomonas aeruginosa infection continues to be a challenge in Latin American countries (LATAM). We synthesize the literature on the use of appropriate initial antibiotic therapy (AIAT) and inappropriate initial antibiotic therapy (IIAT) in P. aeruginosa infections, and the literature on risk factors for acquisition of resistant P. aeruginosa among hospitalized adult patients in LATAM. METHODS: MEDLINE, EMBASE, Cochrane, and LILAC were searched between 2000 and August 2019. Abstracts and full-text articles were screened in duplicate. Random effects meta-analysis was conducted when studies were sufficiently similar. RESULTS: The screening of 165 citations identified through literature search yielded 98 full-text articles that were retrieved and assessed for eligibility, and 19 articles conducted in Brazil (14 articles), Colombia (4 articles), and Cuba (1 article) met the inclusion criteria. Of 19 eligible articles, six articles (840 subjects) examined AIAT compared to IIAT in P. aeruginosa infections; 17 articles (3203 total subjects) examined risk factors for acquisition of resistant P. aeruginosa; and four articles evaluated both. Four of 19 articles were rated low risk of bias and the remaining were deemed unclear or high risk of bias. In meta-analysis, AIAT was associated with lower mortality for P. aeruginosa infections (unadjusted summary OR 0.48, 95% CI 0.28-0.81; I2 = 59%), compared to IIAT and the association with mortality persisted in subgroup meta-analysis by low risk of bias (3 articles; unadjusted summary OR 0.46, 95% CI 0.28-0.81; I2 = 0%). No meta-analysis was performed for studies evaluating risk factors for acquisition of resistant P. aeruginosa as they were not sufficiently similar. Significant risk factors for acquisition of resistant P. aeruginosa included: prior use of antibiotics (11 articles), stay in the intensive care unit (ICU) (3 articles), and comorbidity score (3 articles). Outcomes were graded to be of low strength of evidence owing to unclear or high risk of bias and imprecise estimates. CONCLUSION: Our study highlights the association of AIAT with lower mortality and prior use of antibiotics significantly predicts acquiring resistant P. aeruginosa infections. This review reinforces the need for rigorous and structured antimicrobial stewardship programs in the LATAM region.

Crystal structure and transient dimerization for the FKBP12 protein from the pathogenic fungus Candida auris.

International concern over the recent emergence of Candida auris infections reflects not only its comparative ease of transmission and substantial mortality but the increasing level of resistance observed to all three major classes of antifungal drugs. Diminution in virulence has been reported for a wide range of fungal pathogens when the FK506-binding protein FKBP12 binds to that immunosuppressant drug and the binary complex then inhibits the fungal calcineurin signaling pathway. Structure-based drug design efforts have described modifications of FK506 which modestly reduce virulence for a number of fungal pathogens while also lessening the side effect of suppressing the tissue immunity response in the patient. To aid in such studies, we report the crystal structure of Candida auris FKBP12. As physiological relevance has been proposed for transient homodimerization interactions of distantly related fungal FKBP12 proteins, we report the solution NMR characterization of the homodimerization interactions of the FKBP12 proteins from both Candida auris and Candida glabrata.

1H, 13C, 15 N chemical shift assignments of the FKBP12 protein from the pathogenic fungi Candida auris and Candida glabrata.

Multi-drug resistance is becoming an increasingly severe clinical challenge not only among pathogenic bacteria but among fungal pathogens as well. Drug design is inherently more challenging for the eukaryotic fungi due to their closer evolutionary similarity to humans. The recent rapid expansion in invasive infections throughout the world by Candida auris is of particular concern due to a substantial mortality rate, comparatively facile transmission, and an increasing level of resistance to all three of the major classes of anti-fungal drugs. One promising avenue for the development of an alternative class of anti-fungal agents currently under investigation is for drugs against the FK506-binding protein FKBP12 which, when bound to that drug, inhibits the fungal calcineurin signaling pathway with a resultant diminution in virulence. The specific challenge to this approach is that the homologous human calcineurin pathway functions in controlling the tissue immunity response, so that drug selectivity for the fungal pathway must be designed. To facilitate such efforts, we report the nearly complete backbone and sidechain resonances for the FKBP12 proteins of both Candida auris and clinically significant Candida glabrata fungi.

Prediction of Bond Vector Autocorrelation Functions from Larmor Frequency-Selective Order Parameter Analysis of NMR Relaxation Data.

Protein molecular dynamics interpretation of the standard R1, R2, and heteronuclear NOE relaxation measurements has typically been limited to a single S2 order parameter which is often insufficient to characterize the rich content of these NMR experiments. In the absence of exchange linebroadening, an optimized reduced spectral density analysis of these measurements can yield spectral density values at three distinct frequencies. Surprisingly, these three discrete spectral density values have proven to be sufficient for a Larmor frequency-selective order parameter analysis of the 223 methine and methylene H-C bonds of the B3 domain of Protein G (GB3) to accurately back-calculate the entire curve of the corresponding bond vector autocorrelation functions upon which the NMR relaxation behavior depends. The 13C relaxation values calculated from 2 µs of CHARMM36 simulation trajectories yielded the corresponding autocorrelation functions to an average rmsd of 0.44% with only three bond vectors having rmsd errors slightly greater than 1.0%. Similar quality predictions were obtained using the CHARMM22/CMAP, AMBER ff99SB, and AMBER ff99SB-ILDN force fields. Analogous predictions for the backbone 15N relaxation values were 3-fold more accurate. Excluding seven residues for which either experimental data is lacking or previous MD studies have indicated markedly divergent dynamics predictions, the CHARMM36-derived and experimentally derived 15N relaxation values for the remaining 48 amides of GB3 agree to an average of 0.016, 0.010, and 0.020 for the fast limit (Sf2) and Larmor frequency-selective (SH2 and SN2) order parameters, respectively. In contrast, for a substantial fraction of side chain positions, the statistical uncertainties obtained in the relaxation value predictions from each force field were appreciably less than the much larger differences predicted among these force fields, indicating a significant opportunity for experimental NMR relaxation measurements to provide structurally interpretable guidance for further optimizing the prediction of protein dynamics.

Quantifying protein dynamics in the ps-ns time regime by NMR relaxation.

Both 15N chemical shift anisotropy (CSA) and sufficiently rapid exchange linebroadening transitions exhibit relaxation contributions that are proportional to the square of the magnetic field. Deconvoluting these contributions is further complicated by residue-dependent variations in protein amide 15N CSA values which have proven difficult to accurately measure. Exploiting recently reported improvements for the implementation of T1 and T1ρ experiments, field strength-dependent studies have been carried out on the B3 domain of protein G (GB3) as well as on the immunophilin FKBP12 and a H87V variant of that protein in which the major conformational exchange linebroadening transition is suppressed. By applying a zero frequency spectral density rescaling analysis to the relaxation data collected at magnetic fields from 500 to 900 MHz 1H, differential residue-specific 15N CSA values have been obtained for GB3 which correlate with those derived from solid state and liquid crystalline NMR measurements to a level similar to the correlation among those previously reported studies. Application of this analysis protocol to FKBP12 demonstrated an efficient quantitation of both weak exchange linebroadening contributions and differential residue-specific 15N CSA values. Experimental access to such differential residue-specific 15N CSA values should significantly facilitate more accurate comparisons with molecular dynamics simulations of protein motion that occurs within the timeframe of global molecular tumbling.

Conformational Dynamics in FKBP Domains: Relevance to Molecular Signaling and Drug Design.

Among the 22 FKBP domains in the human genome, FKBP12.6 and the first FKBP domains (FK1) of FKBP51 and FKBP52 are evolutionarily and structurally most similar to the archetypical FKBP12. As such, the development of inhibitors with selectivity among these four FKBP domains poses a significant challenge for structure-based design. The pleiotropic effects of these FKBP domains in a range of signaling processes such as the regulation of ryanodine receptor calcium channels by FKBP12 and FKBP12.6 and steroid receptor regulation by the FK1 domains of FKBP51 and FKBP52 amply justify the efforts to develop selective therapies. In contrast to their close structural similarities, these four FKBP domains exhibit a substantial diversity in their conformational flexibility. A number of distinct conformational transitions have been characterized for FKBP12 spanning timeframes from 20 s to 10 ns and in each case these dynamics have been shown to markedly differ from the conformational behavior for one or more of the other three FKBP domains. Protein flexibilitybased inhibitor design could draw upon the transitions that are significantly populated in only one of the targeted proteins. Both the similarities and differences among these four proteins valuably inform the understanding of how dynamical effects propagate across the FKBP domains as well as potentially how such intramolecular transitions might couple to the larger scale transitions that are central to the signaling complexes in which these FKBP domains function.

Coupling of Conformational Transitions in the N-terminal Domain of the 51-kDa FK506-binding Protein (FKBP51) Near Its Site of Interaction with the Steroid Receptor Proteins.

Interchanging Leu-119 for Pro-119 at the tip of the ß4-ß5 loop in the first FK506 binding domain (FK1) of the FKBP51 and FKBP52 proteins, respectively, has been reported to largely reverse the inhibitory (FKBP51) or stimulatory (FKBP52) effects of these co-chaperones on the transcriptional activity of glucocorticoid and androgen receptor-protein complexes. Previous NMR relaxation studies have identified exchange line broadening, indicative of submillisecond conformational motion, throughout the ß4-ß5 loop in the FK1 domain of FKBP51, which are suppressed by the FKBP52-like L119P substitution. This substitution also attenuates exchange line broadening in the underlying ß2 and ß3a strands that is centered near a bifurcated main chain hydrogen bond interaction between these two strands. The present study demonstrates that these exchange line broadening effects arise from two distinct coupled conformational transitions, and the transition within the ß2 and ß3a strands samples a transient conformation that resembles the crystal structures of the selectively inhibited FK1 domain of FKBP51 recently reported. Although the crystal structures for their series of inhibitors were interpreted as evidence for an induced fit mechanism of association, the presence of a similar conformation being significantly populated in the unliganded FKBP51 domain is more consistent with a conformational selection binding process. The contrastingly reduced conformational plasticity of the corresponding FK1 domain of FKBP52 is consistent with the current model in which FKBP51 binds to both the apo- and hormone-bound forms of the steroid receptor to modulate its affinity for ligand, whereas FKBP52 binds selectively to the latter state.

Statistical allosteric coupling to the active site indole ring flip equilibria in the FK506-binding domain.

In solution, the Trp 59 indole ring at the base of the active site cleft in the FKBP domain protein FKBP12 is rotated by ~90° at a population level of 20%, relative to its canonical crystallographic orientation. NMR measurements on the homologous FK1 domains of human FKBP51 and FKBP52 indicate no observable indole ring flip conformation, while the V101I variant of FKBP12 decreases the population having a perpendicular indole orientation by 10-fold. A set of three parallel 400 ns CHARMM27 molecular simulations for both wild type FKBP12 and the V101I variant examined how this ring flip might be energetically coupled to a transition of the Glu 60 sidechain which interacts with the backbone of the 50's loop located ~12 Å from the indole nitrogen. Analysis of the transition matrix for the local dynamics of the Glu 60 sidechain, the Trp 59 sidechain, and of the structurally interposed α-helix hydrogen bonding pattern yielded a statistical allosteric coupling of 10 kJ/mol with negligible concerted dynamical coupling for the transitions of the two sidechains.

Differential conformational dynamics in the closely homologous FK506-binding domains of FKBP51 and FKBP52.

As co-chaperones of Hsp90 (heat-shock protein 90), FKBP51 (FK506-binding protein of 51 kDa) and FKBP52 (FK506-binding protein of 52 kDa) act as antagonists in regulating the hormone affinity and nuclear transport of steroid receptor complexes. Exchange of Leu119 in FKBP51 for Pro119 in FKBP52 has been shown to largely reverse the steroid receptor activities of FKBP51 and FKBP52. To examine whether differences in conformational dynamics/plasticity might correlate with changes in the reported receptor activities, 15N-NMR relaxation measurements were carried out on the N-terminal FKBP domains of FKBP51 and FKBP52 as well as their residue-swapped variants. Both proteins exhibit a similar pattern of motion in the picosecond-nanosecond timeframe as well as a small degree of 15N line-broadening, indicative of motion in the microsecond-millisecond timeframe, in the ß3a strand of the central sheet. Only the FKBP51 domain exhibits much larger line-broadening in the adjacent ß3 bulge (40's loop of FKBP12) and throughout the long ß4-ß5 loop (80's loop of FKBP12). The L119P mutation at the tip of the ß4-ß5 loop completely suppressed the line-broadening in this loop while partially suppressing the line-broadening in the neighbouring ß2 and ß3a strands. The complementary P119L and P119L/P124S variants of FKBP52 yielded similar patterns of line-broadening for the ß4-ß5 loop as that for FKBP51, although only 20% and 60% as intense respectively. However, despite the close structural similarity in the packing interactions between the ß4-ß5 loop and the ß3a strand for FKBP51 and FKBP52, the line-broadening in the ß3a strand is unaffected by the P119L or P119L/P124S mutations in FKBP52.

Crystal structure and conformational flexibility of the unligated FK506-binding protein FKBP12.6.

The primary known physiological function of FKBP12.6 involves its role in regulating the RyR2 isoform of ryanodine receptor Ca(2+) channels in cardiac muscle, pancreatic ß islets and the central nervous system. With only a single previously reported X-ray structure of FKBP12.6, bound to the immunosuppressant rapamycin, structural inferences for this protein have been drawn from the more extensive studies of the homologous FKBP12. X-ray structures at 1.70 and 1.90 Šresolution from P21 and P3121 crystal forms are reported for an unligated cysteine-free variant of FKBP12.6 which exhibit a notable diversity of conformations. In one monomer from the P3121 crystal form, the aromatic ring of Phe59 at the base of the active site is rotated perpendicular to its typical orientation, generating a steric conflict for the immunosuppressant-binding mode. The peptide unit linking Gly89 and Val90 at the tip of the protein-recognition `80s loop' is flipped in the P21 crystal form. Unlike the >30 reported FKBP12 structures, the backbone conformation of this loop closely follows that of the first FKBP domain of FKBP51. The NMR resonances for 21 backbone amides of FKBP12.6 are doubled, corresponding to a slow conformational transition centered near the tip of the 80s loop, as recently reported for 31 amides of FKBP12. The comparative absence of doubling for residues along the opposite face of the active-site pocket in FKBP12.6 may in part reflect attenuated structural coupling owing to increased conformational plasticity around the Phe59 ring.

Structural basis of conformational transitions in the active site and 80's loop in the FK506-binding protein FKBP12.

The extensive set of NMR doublings exhibited by the immunophilin FKBP12 (FK506-binding protein 12) arose from a slow transition to the cis-peptide configuration at Gly89 near the tip of the 80's loop, the site for numerous protein-recognition interactions for both FKBP12 and other FKBP domain proteins. The 80's loop also exhibited linebroadening, indicative of microsecond to millisecond conformational dynamics, but only in the trans-peptide state. The G89A variant shifted the trans-cis peptide equilibrium from 88:12 to 33:67, whereas a proline residue substitution induced fully the cis-peptide configuration. The 80's loop conformation in the G89P crystal structure at 1.50 Å resolution differed from wild-type FKBP12 primarily at residues 88, 89 and 90, and it closely resembled that reported for FKBP52. Structure-based chemical-shift predictions indicated that the microsecond to millisecond dynamics in the 80's loop probably arose from a concerted main chain (ψ88 and ϕ89) torsion angle transition. The indole side chain of Trp59 at the base of the active-site cleft was reoriented ~90o and the adjacent backbone was shifted in the G89P crystal structure. NOE analysis of wild-type FKBP12 demonstrated that this indole populates the perpendicular orientation at 20%. The 15N relaxation analysis was consistent with the indole reorientation occurring in the nanosecond timeframe. Recollection of the G89P crystal data at 1.20 Å resolution revealed a weaker wild-type-like orientation for the indole ring. Differences in the residues that underlie the Trp59 indole ring and altered interactions linking the 50's loop to the active site suggested that reorientation of this ring may be disfavoured in the other six members of the FKBP domain family that bear this active-site tryptophan residue.

Analysing the visible conformational substates of the FK506-binding protein FKBP12.

The 1H-15N 2D NMR correlation spectrum of the widely studied FK506-binding protein FKBP12 (FK506-binding protein of 12 kDa) contains previously unreported peak doublings for at least 31 residues that arise from a minor conformational state (12% of total) which exchanges with the major conformation with a time constant of 3.0 s at 43°C. The largest differences in chemical shift occur for the 80's loop that forms critical recognition interactions with many of the protein partners for the FKBP family. The residues exhibiting doubling extend into the adjacent strands of the ß-sheet, across the active site to the α-helix and into the 50's loop. Each of the seven proline residues adopts a trans-peptide linkage in both the major and minor conformations, indicating that this slow transition is not the result of prolyl isomerization. Many of the residues exhibiting resonance doubling also participate in conformational line-broadening transition(s) that occur ~105-fold more rapidly, proposed previously to arise from a single global process. The 1.70 Å (1 Å=0.1 nm) resolution X-ray structure of the H87V variant is strikingly similar to that of FKBP12, yet this substitution quenches the slow conformational transition throughout the protein while quenching the line-broadening transition for residues near the 80's loop. Line-broadening was also decreased for the residues in the α-helix and 50's loop, whereas line-broadening in the 40's loop was unaffected. The K44V mutation selectively reduces the line-broadening in the 40's loop, verifying that at least three distinct conformational transitions underlie the line-broadening processes of FKBP12.

Assessing the chemical accuracy of protein structures via peptide acidity.

Although the protein native state is a Boltzmann conformational ensemble, practical applications often require a representative model from the most populated region of that distribution. The acidity of the backbone amides, as reflected in hydrogen exchange rates, is exquisitely sensitive to the surrounding charge and dielectric volume distribution. For each of four proteins, three independently determined X-ray structures of differing crystallographic resolution were used to predict exchange for the static solvent-exposed amide hydrogens. The average correlation coefficients range from 0.74 for ubiquitin to 0.93 for Pyrococcus furiosus rubredoxin, reflecting the larger range of experimental exchange rates exhibited by the latter protein. The exchange prediction errors modestly correlate with the crystallographic resolution. MODELLER 9v6-derived homology models at ~60% sequence identity (36% identity for chymotrypsin inhibitor CI2) yielded correlation coefficients that are ~0.1 smaller than for the cognate X-ray structures. The most recently deposited NOE-based ubiquitin structure and the original NMR structure of CI2 fail to provide statistically significant predictions of hydrogen exchange. However, the more recent RECOORD refinement study of CI2 yielded predictions comparable to the X-ray and homology model-based analyses.

Experimentally assessing molecular dynamics sampling of the protein native state conformational distribution.

The acute sensitivity to conformation exhibited by amide hydrogen exchange reactivity provides a valuable test for the physical accuracy of model ensembles developed to represent the Boltzmann distribution of the protein native state. A number of molecular dynamics studies of ubiquitin have predicted a well-populated transition in the tight turn immediately preceding the primary site of proteasome-directed polyubiquitylation Lys 48. Amide exchange reactivity analysis demonstrates that this transition is 10(3)-fold rarer than these predictions. More strikingly, for the most populated novel conformational basin predicted from a recent 1 ms MD simulation of bovine pancreatic trypsin inhibitor (at 13% of total), experimental hydrogen exchange data indicates a population below 10(-6). The most sophisticated efforts to directly incorporate experimental constraints into the derivation of model protein ensembles have been applied to ubiquitin, as illustrated by three recently deposited studies (PDB codes 2NR2, 2K39 and 2KOX2K392KOX). Utilizing the extensive set of experimental NOE constraints, each of these three ensembles yields a modestly more accurate prediction of the exchange rates for the highly exposed amides than does a standard unconstrained molecular simulation. However, for the less frequently exposed amide hydrogens, the 2NR2 ensemble offers no improvement in rate predictions as compared to the unconstrained MD ensemble. The other two NMR-constrained ensembles performed markedly worse, either underestimating (2KOX) or overestimating (2K39) the extent of conformational diversity.