Protein NMR Structures Refined with Rosetta Have Higher Accuracy Relative to Corresponding X‑ray Crystal Structures

We have found that refinement of protein NMR structures using Rosetta with experimental NMR restraints yields more accurate protein NMR structures than those that have been deposited in the PDB using standard refinement protocols. Using 40 pairs of NMR and X-ray crystal structures determined by the Northeast Structural Genomics Consortium, for proteins ranging in size from 5–22 kDa, restrained Rosetta refined structures fit better to the raw experimental data, are in better agreement with their X-ray counterparts, and have better phasing power compared to conventionally determined NMR structures. For 37 proteins for which NMR ensembles were available and which had similar structures in solution and in the crystal, all of the restrained Rosetta refined NMR structures were sufficiently accurate to be used for solving the corresponding X-ray crystal structures by molecular replacement. The protocol for restrained refinement of protein NMR structures was also compared with restrained CS-Rosetta calculations. For proteins smaller than 10 kDa, restrained CS-Rosetta, starting from extended conformations, provides slightly more accurate structures, while for proteins in the size range of 10–25 kDa the less CPU intensive restrained Rosetta refinement protocols provided equally or more accurate structures. The restrained Rosetta protocols described here can improve the accuracy of protein NMR structures and should find broad and general for studies of protein structure and function

Inhibitor Bound Dengue NS2B-NS3pro Reveals Multiple Dynamic Binding Modes

Dengue virus poses a significant global health threat as the source of increasingly deleterious dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. As no specific antiviral treatment exists for dengue infection, considerable effort is being applied to discover therapies and drugs for maintenance and prevention of these afflictions. The virus is primarily transmitted by mosquitoes, and infection occurs following viral endocytosis by host cells. Upon entering the cell, viral RNA is translated into a large multisubunit polyprotein which is post-translationally cleaved into mature, structural and nonstructural (NS) proteins. The viral genome encodes the enzyme to carry out cleavage of the large polyprotein, specifically the NS2B-NS3pro cofactor-protease complexa target of high interest for drug design. One class of recently discovered NS2B-NS3pro inhibitors is the substrate-based trifluoromethyl ketone containing peptides. These compounds interact covalently with the active site Ser135 via a hemiketal adduct. A detailed picture of the intermolecular protease/inhibitor interactions of the hemiketal adduct is crucial for rational drug design. We demonstrate, through the use of protein- and ligand-detected solution-state ¹⁹F and ¹H NMR methods, an unanticipated multibinding mode behavior of a representative of this class of inhibitors to dengue NS2B-NS3pro. Our results illustrate the highly dynamic nature of both the covalently bound ligand and protease protein structure, and the need to consider these dynamics when designing future inhibitors in this class

ATPase Active-Site Electrostatic Interactions Control the Global Conformation of the 100 kDa SecA Translocase

SecA is an intensively studied mechanoenzyme that uses ATP hydrolysis to drive processive extrusion of secreted proteins through a protein-conducting channel in the cytoplasmic membrane of eubacteria. The ATPase motor of SecA is strongly homologous to that in DEAD-box RNA helicases. It remains unclear how local chemical events in its ATPase active site control the overall conformation of an ∼100 kDa multidomain enzyme and drive protein transport. In this paper, we use biophysical methods to establish that a single electrostatic charge in the ATPase active site controls the global conformation of SecA. The enzyme undergoes an ATP-modulated endothermic conformational transition (ECT) believed to involve similar structural mechanics to the protein transport reaction. We have characterized the effects of an isosteric glutamate-to-glutamine mutation in the catalytic base, a mutation which mimics the immediate electrostatic consequences of ATP hydrolysis in the active site. Calorimetric studies demonstrate that this mutation facilitates the ECT in Escherichia coli SecA and triggers it completely in Bacillus subtilis SecA. Consistent with the substantial increase in entropy observed in the course of the ECT, hydrogen–deuterium exchange mass spectrometry demonstrates that it increases protein backbone dynamics in domain–domain interfaces at remote locations from the ATPase active site. The catalytic glutamate is one of ∼250 charged amino acids in SecA, and yet neutralization of its side chain charge is sufficient to trigger a global order–disorder transition in this 100 kDa enzyme. The intricate network of structural interactions mediating this effect couples local electrostatic changes during ATP hydrolysis to global conformational and dynamic changes in SecA. This network forms the foundation of the allosteric mechanochemistry that efficiently harnesses the chemical energy stored in ATP to drive complex mechanical processes

Analysis of the Structural Quality of the CASD-NMR 2013 Entries

We performed a comprehensive structure validation of both automated and manually generated structures of the 10 targets of the CASD-NMR-2013 effort. We established that automated structure determination protocols are capable of reliably producing structures of comparable accuracy and quality to those generated by a skilled researcher, at least for small, single domain proteins such as the ten targets tested. The most robust results appear to be obtained when NOESY peak lists are used either as the primary input data or to augment Chemical Shift (CS) data without the need to manually filter such lists. A detailed analysis of the long-range NOE restraints generated by the different programs from the same data showed a surprisingly low degree of overlap. Additionally, we found that there was no significant correlation between the extent of the NOE restraint overlap and the accuracy of the structure. This result was surprising given the importance of NOE data in producing good quality structures. We suggest that this could be explained by the information redundancy present in NOEs between atoms contained within a fixed covalent network

Structure of the BfR192 exopolyphosphatase-related protein showing the two domains and highlighting the cleft containing the sodium, potassium and four phosphate ions.

<p>Structure of the BfR192 exopolyphosphatase-related protein showing the two domains and highlighting the cleft containing the sodium, potassium and four phosphate ions.</p

Enzyme Engineering Based on X‑ray Structures and Kinetic Profiling of Substrate Libraries: Alcohol Dehydrogenases for Stereospecific Synthesis of a Broad Range of Chiral Alcohols

The narrow substrate scope of naturally occurring alcohol dehydrogenases (ADHs) greatly limits the enzymatic synthesis of important chiral alcohols. On the basis of X-ray crystal structures and kinetic profiling of a substrate library, we engineered variants of the stereospecific alcohol dehydrogenase from Candida parapsilopsis. This resulted in a set of four mutant enzymes which enable the asymmetric reduction of a broad range of prochiral ketones, including valuable pharmaceuticals and fine chemicals. The engineering strategy of this study paves the way for creating additional ADHs tailored for production of complex chiral alcohols

Each of these heatmaps represents a metric comparison between two consecutive generations of a screen from the Hauptman-Woodward Medical Research Institute [8].

<p>The screens have 1,536 cocktails, and the heatmaps can be viewed as overlays of the 1,536-well plate in which these screens reside, with the colors of each block representing the metric difference between the successive cocktails in that particular location on the plate. Each square unit of color corresponds to the comparison between the cocktails in the successive generations in that location on the plate. In the top, the C6 metric is used while the <i>CD_coeff</i> is shown below. Both metrics were able to highlight two rows of cocktails that were altered considerably between generations 8 and 8A, in the form of a line of darker wells in the lower third of figures. The C6 metric, however, identified that cocktails outside of these two rows were slightly different, when they were actually identical. This discrepancy most likely arises from the C6 metric's use of penalties in its PEG and salt terms.</p

Regions of crystallization space where hits for BfR192 were found.

<p>Out of the 28 clusters, 11 were identified containing at least 1 crystal hit. The full list is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100782#pone-0100782-t004" target="_blank">Table 4</a>.</p

Cocktails that produced visually recognizable crystals in the clusters identified in Figure 7.

<p>Cocktails that produced visually recognizable crystals in the clusters identified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100782#pone-0100782-g007" target="_blank">Figure 7</a>.</p

Determination of the Structures of Symmetric Protein Oligomers from NMR Chemical Shifts and Residual Dipolar Couplings

Symmetric protein dimers, trimers, and higher-order cyclic oligomers play key roles in many biological processes. However, structural studies of oligomeric systems by solution NMR can be difficult due to slow tumbling of the system and the difficulty in identifying NOE interactions across protein interfaces. Here, we present an automated method (RosettaOligomers) for determining the solution structures of oligomeric systems using only chemical shifts, sparse NOEs, and domain orientation restraints from residual dipolar couplings (RDCs) without a need for a previously determined structure of the monomeric subunit. The method integrates previously developed Rosetta protocols for solving the structures of monomeric proteins using sparse NMR data and for predicting the structures of both nonintertwined and intertwined symmetric oligomers. We illustrated the performance of the method using a benchmark set of nine protein dimers, one trimer, and one tetramer with available experimental data and various interface topologies. The final converged structures are found to be in good agreement with both experimental data and previously published high-resolution structures. The new approach is more readily applicable to large oligomeric systems than conventional structure-determination protocols, which often require a large number of NOEs, and will likely become increasingly relevant as more high-molecular weight systems are studied by NMR