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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Peptide conformer acidity analysis of protein flexibility monitored by hydrogen exchange.

The amide hydrogens that are exposed to solvent in the high-resolution X-ray structures of ubiquitin, FK506-binding protein, chymotrypsin inhibitor 2, and rubredoxin span a billion-fold range in hydroxide-catalyzed exchange rates which are predictable by continuum dielectric methods. To facilitate analysis of transiently accessible amides, the hydroxide-catalyzed rate constants for every backbone amide of ubiquitin were determined under near physiological conditions. With the previously reported NMR-restrained molecular dynamics ensembles of ubiquitin (PDB codes 2NR2 and 2K39 ) used as representations of the Boltzmann-weighted conformational distribution, nearly all of the exchange rates for the highly exposed amides were more accurately predicted than by use of the high-resolution X-ray structure. More strikingly, predictions for the amide hydrogens of the NMR relaxation-restrained ensemble that become exposed to solvent in more than one but less than half of the 144 protein conformations in this ensemble were almost as accurate. In marked contrast, the exchange rates for many of the analogous amides in the residual dipolar coupling-restrained ubiquitin ensemble are substantially overestimated, as was particularly evident for the Ile 44 to Lys 48 segment which constitutes the primary interaction site for the proteasome targeting enzymes involved in polyubiquitylation. For both ensembles, "excited state" conformers in this active site region having markedly elevated peptide acidities are represented at a population level that is 10(2) to 10(3) above what can exist in the Boltzmann distribution of protein conformations. These results indicate how a chemically consistent interpretation of amide hydrogen exchange can provide insight into both the population and the detailed structure of transient protein conformations.

Polarization and polarizability assessed by protein amide acidity.

Hydroxide-catalyzed exchange rate constants were determined for those amides of FK506-binding protein (FKBP12), ubiquitin, and chymotrypsin inhibitor 2 (CI2) that are solvent-accessible in the high-resolution X-ray structures. When combined with previous hydrogen exchange results for the rubredoxin from Pyrococcus furiosus, the acidity of these amides was calculated by continuum dielectric methods as a function of the nonpolarizable electrostatic parameter set, internal dielectric, and the charge distribution of the peptide anion. The CHARMM22 parameter set with an internal dielectric value of 3 and an ab initio-derived anion charge distribution yielded an rmsd value of 7 for the 56 amide exchange rate constants ranging from 10(0.67) to 10(9.0) M(-1) s(-1). The OPLS-AA parameter set yielded comparably robust predictions, while that of PARSE, AMBER parm99, and AMBER ff03 performed more poorly. The small value for the optimal internal dielectric, combined with the brief lifetime of the peptide anion intermediate and the uniformity of the correlation between predicted and observed amide acidities, is consistent with electronic polarizability providing the dominant contribution to dielectric shielding. By construction, nonpolarizable force fields do not model electric field attenuation by electronic polarizability. Accurate prediction of the total electrostatic energy by such force fields necessitates the hyperpolarization of the atomic charge values in order to match the average electric field energy density (1/2)epsilon(tau)E(2)(tau) when epsilon(tau) is set to the in vacuo dielectric value of 1. The resulting predictions of the experimental hydrogen exchange data demonstrate the substantial systematic errors in the predicted electrostatic potential that can arise when dielectric shielding due to electronic polarizability is neglected.

NMR analysis of native-state protein conformational flexibility by hydrogen exchange.

The rate of hydrogen exchange for the most protected amides of a protein is widely used to provide an estimate of global conformational stability by analyzing the exchange kinetics in the unfolded state in terms of model peptide exchange rates. The exchange behavior of the other amides of the protein which do not exchange via a global unfolding mechanism can provide insight into the smaller-scale conformational transitions that facilitate access to solvent as required for the exchange reaction. However, since the residual tertiary structure in the exchange-competent conformation can modulate the chemistry of the exchange reaction, equilibrium values estimated from normalization with model peptide rates are open to question. To overcome this limitation, the most robust approaches utilize differential analyses as a function of experimental variables such as denaturant concentration, temperature, pH, and mutational variation. Practical aspects of these various differential analysis techniques are considered with illustrations drawn from the literature.

A billion-fold range in acidity for the solvent-exposed amides of Pyrococcus furiosus rubredoxin.

The exchange rates of the static solvent-accessible amide hydrogens of Pyrococcus furiosus rubredoxin range from near the diffusion-limited rate to a billion-fold slower for the non-hydrogen-bonded Val 38 (eubacterial numbering). Hydrogen exchange directly monitors the kinetic acidity of the peptide nitrogen. Electrostatic solvation free energies were calculated by Poisson-Boltzmann methods for the individual peptide anions that form during the hydroxide-catalyzed exchange reaction to examine how well the predicted thermodynamic acidities match the experimentally determined kinetic acidities. With the exception of the Ile 12 amide, the differential exchange rate constant for each solvent-exposed amide proton that is not hydrogen bonded to a backbone carbonyl can be predicted within a factor of 6 (10 (0.78)) root-mean-square deviation (rmsd) using the CHARMM22 electrostatic parameter set and an internal dielectric value of 3. Under equivalent conditions, the PARSE parameter set yields a larger rmsd value of 1.28 pH units, while the AMBER parm99 parameter set resulted in a considerably poorer correlation. Either increasing the internal dielectric value to 4 or reducing it to a value of 2 significantly degrades the quality of the prediction. Assigning the excess charge of the peptide anion equally between the peptide nitrogen and the carbonyl oxygen also reduces the correlation to the experimental data. These continuum electrostatic calculations were further analyzed to characterize the specific structural elements that appear to be responsible for the wide range of peptide acidities observed for these solvent-exposed amides. The striking heterogeneity in the potential at sites along the protein-solvent interface should prove germane to the ongoing challenge of quantifying the contribution that electrostatic interactions make to the catalytic acceleration achieved by enzymes.

Electrostatic stabilization and general base catalysis in the active site of the human protein disulfide isomerase a domain monitored by hydrogen exchange.

The nucleophilic Cys36 thiol of the human protein disulfide isomerase a domain is positioned over the N terminus of the alpha(2) helix. Amides in the active site exhibit diffusion-limited, hydroxide-catalyzed exchange, indicating that the local positive electrostatic potential decreases the pK value for peptide anion formation by at least 2 units so as to equal or exceed the acidity of water. In stark contrast to the pH dependence of exchange for simple peptides, the His38 amide in the reduced enzyme exhibits a maximum rate of exchange at pH 5 due to efficient general base catalysis by the neutral imidazole of its own side chain and suppression of its exchange by the ionization of the Cys36 thiol. Ionization of this thiol and deprotonation of the His38 side chain suppress the Cys39 amide hydroxide-catalyzed exchange by a million-fold. The electrostatic potential within the active site monitored by these exchange experiments provides a means of stabilizing the two distinct transition states that lead to substrate reduction and oxidation. Molecular modeling offers a role for the conserved Arg103 in coordinating the oxidative transition-state complex, thus providing further support for mechanisms of disulfide isomerization that utilize enzymatic catalysis at each step of the overall reaction.

NMR and X-ray analysis of structural additivity in metal binding site-swapped hybrids of rubredoxin.

BACKGROUND: Chimeric hybrids derived from the rubredoxins of Pyrococcus furiosus (Pf) and Clostridium pasteurianum (Cp) provide a robust system for the characterization of protein conformational stability and dynamics in a differential mode. Interchange of the seven nonconserved residues of the metal binding site between the Pf and Cp rubredoxins yields a complementary pair of hybrids, for which the sum of the thermodynamic stabilities is equal to the sum for the parental proteins. Furthermore, the increase in amide hydrogen exchange rates for the hyperthermophile-derived metal binding site hybrid is faithfully mirrored by a corresponding decrease for the complementary hybrid that is derived from the less thermostable rubredoxin, indicating a degree of additivity in the conformational fluctuations that underlie these exchange reactions. RESULTS: Initial NMR studies indicated that the structures of the two complementary hybrids closely resemble "cut-and-paste" models derived from the parental Pf and Cp rubredoxins. This protein system offers a robust opportunity to characterize differences in solution structure, permitting the quantitative NMR chemical shift and NOE peak intensity data to be analyzed without recourse to the conventional conversion of experimental NOE peak intensities into distance restraints. The intensities for 1573 of the 1652 well-resolved NOE crosspeaks from the hybrid rubredoxins were statistically indistinguishable from the intensities of the corresponding parental crosspeaks, to within the baseplane noise level of these high sensitivity data sets. The differences in intensity for the remaining 79 NOE crosspeaks were directly ascribable to localized dynamical processes. Subsequent X-ray analysis of the metal binding site-swapped hybrids, to resolution limits of 0.79 A and 1.04 A, demonstrated that the backbone and sidechain heavy atoms in the NMR-derived structures lie within the range of structural variability exhibited among the individual molecules in the crystallographic asymmetric unit (approximately 0.3 A), indicating consistency with the "cut-and-paste" structuring of the hybrid rubredoxins in both crystal and solution. CONCLUSION: Each of the significant energetic interactions in the metal binding site-swapped hybrids appears to exhibit a 1-to-1 correspondence with the interactions present in the corresponding parental rubredoxin structure, thus providing a structural basis for the observed additivity in conformational stability and dynamics. The congruence of these X-ray and NMR experimental data offers additional support for the interpretation that the conventional treatment of NOE distance restraints contributes substantially to the systematic differences that are commonly reported between NMR- and X-ray-derived protein structures.

Residue cluster additivity of thermodynamic stability in the hydrophobic core of mesophile vs. hyperthermophile rubredoxins.

The branched sidechain residues 24 and 33 in the hydrophobic core of rubredoxin differ between the Clostridium pasteurianum (Cp) and Pyrococcus furiosus (Pf) sequences. Their X-ray structures indicate that these two sidechains are in van der Waals contact with each other, while neither appears to significantly interact with the other nonconserved residues. The simultaneous interchange of residues 24 and 33 between the Cp and Pf rubredoxin sequences yield a complementary pair of hybrid proteins for which the sum of their thermodynamic stabilities equals that of the parental rubredoxins. The 1.2 kcal/mol change arising from this two residues interchange accounts for 21% of the differential thermodynamic stability between the mesophile and hyperthermophile proteins. The additional interchange of the sole nonconserved aromatic residue in the hydrophobic core yields a 0.78 kcal/mol deviation from thermodynamic additivity.