Thymidylate kinase (TMK) from Candida albicans (CaTMK) contains a unique 15 residue insert, the CaLoop, that is not found on other TMKs. CaTMK is proficient at phosphorylating deoxyuridine monophosphate (dUMP), showing a rate 6-fold higher than TMP. It has been shown that deletion of the CaLoop reduces the activity towards dUMP by 19-fold, but has only a modest 4-fold decrease in activity towards TMP. The molecular dynamics calculations presented here show that the increased activity towards dUMP is due to an increase in flexibility and correlated motions of the protein that allows the enzyme-dUMP complex to more readily approach a catalytically competent state. Deletion of the CaLoop allows the dUMP-enzyme complex to adopt catalytically non-functional conformations. In contrast, TMP stabilizes the deletion such that it remains in a functional conformation that is similar to the conformation of the original enzyme.

Alongside inorganic materials, water, and air, soil organic matter (SOM) is one of the major components of soil and has tremendous influence on the environment given its vital role in the carbon cycle. Many soil dwelling organisms like plants, fungi and bacteria excrete proteins, whose interaction with SOM is poorly understood on an atomistic level. In this study, molecular dynamics simulations were used to investigate selected proteins in soil models of different complexity from simple co-solvent molecules to Leonardite humic acids (LHA). We analyzed the proteins in terms of their structural stability, the nature and strength of the interactions with their surroundings, as well as their aggregation behavior. Upon insertion of proteins in complex SOM models, their structural stability decreased, although no unfolding or disruption of secondary structure was observed. The interactions of proteins and SOM were primarily governed by electrostatic forces, often in form of hydrogen bonds. However, also weaker van der Waals forces made a significant contribution to the total interaction energies. Moreover, we showed that even though the molecular structure and size of SOM molecules varied, the functional groups of SOM ordered around the protein in a similar pattern. Finally, the number of aggregates formed by proteins and SOM molecules was shown to be primarily proportional to the size of the latter. Strikingly, for varying protein net charges no changes in the formation of aggregates with the strongly negatively charged LHA were observed.

Carbohydrates play a major role in infection strategies of various enteric pathogens. In Campylobacter jejuni, the most common cause of gastroenteritis, uniquely modified heptoses found in surface carbohydrates are synthesized by specific pathways. Owing to the importance of such pathways for the infectious potential of pathogens and/or their virulence, these biosynthesis pathways present potential targets for therapeutic intervention. Here, we determined the crystal structure of GDP-6-OMe-4-keto-L-xylo-heptose reductase (MlghC), an enzyme within the L-gluco-heptose synthesis pathway of C. jejuni strain NCTC 11168. This enzyme lacks the canonical tyrosine residue of the conserved catalytic Ser-Lys-Tyr triad commonly found among functionally related reductases. Despite adopting the overall two-domain fold shared with other short-chain dehydrogenase/reductase family members, subtle structural differences in the interface between the cofactor- and substrate-binding domains explain the absence of epimerase activity and different substrate specificity of this reductase. Modeling of the product-bound complex based on the crystal structure presented here suggests that a tyrosine residue unique to MlghC replaces the missing canonical residue of the catalytic triad.

Small heat shock proteins (sHSPs) are ATP-independent molecular chaperones with low molecular weight that prevent the aggregation of proteins during stress conditions and maintain protein homeostasis in the cell. sHSPs exist in dynamic equilibrium as a mixture of oligomers of various sizes with a constant exchange of subunits between them. Many sHSPs form cage-like assemblies that may dissociate into smaller oligomers during stress conditions. We carried out the functional and structural characterization of a small heat shock protein, HSP18.5, from Entamoeba histolytica (EhHSP18.5). It showed a pH-dependent change in its oligomeric state, which varied from a tetramer to larger than 48-mer. EhHSP18.5 protected Nde I and lysozyme substrates from temperature and chemical stresses, respectively. The crystal structure of EhHSP18.5 was determined at a resolution of 3.28 Å in C2221 cell with four subunits in the asymmetric unit forming two non-metazoan sHSP-type dimers. Unlike the reported cage-like structures, EhHSP18.5 formed a network of linear chains of molecules in the crystal. Instead of a single [IV]-X-[IV] motif, EhHSP18.5 has two overlapping I/V-X-I/V sequences at the C-terminus giving rise to novel interactions between the dimers. Negative staining Electron Microscopy images of EhHSP18.5 showed the presence of multiple oligomers: closed structures of various sizes and long tube-like structures.

MOTIVATION: The assignment of protein secondary structure elements (SSEs) underpins structural analysis and prediction. The backbone of a protein could be adequately represented using a pc-polyline that passes through the centers of its peptide planes. One salient feature of pc-polyline representation is that the secondary structure of a protein becomes recognizable in a matrix whose elements are the pairwise distances between two peptide plane centers. Thus, a pc-polyline could in turn be used to assign SSEs. RESULTS: Using convolutional neural network (CNN) here we confirm that a pc-polyline indeed contains enough information for it to be used for the accurate assignments of the six SSE types: α-helix, β-sheet, β-bulge, 310 -helix, turn and loop. The applications to three large data sets show that the assignments by our CNN-based p2psse program agree very well with those by dssp, stride and quite well with those by five other programs. The analyses of their SSE assignments raise some general questions about the characterizations of protein secondary structure. In particular the analyses illustrate the difficulty with giving a quantitative and consistent definition for each of the six SSE types especially for 310 -helix, β-bulge, turn or loop in terms of either backbone H-bond patterns, or backbone dihedral angles, or Cα -polyline or pc-polyline. The difficulty suggests that the SSE space though being dominated by the regions for the six SSE types is to a certain degree continuous. AVAILABILITY: The program is available at https://github.com/wlincong/p2pSSE.

Light-oxygen-voltage (LOV) domains are widespread photosensory modules that can be used in fluorescence microscopy, optogenetics and controlled production of reactive oxygen species. All of the currently known LOV domains have absorption maxima in the range of ~440 to ~450 nm, and it is not clear whether they can be shifted significantly using mutations. Here, we have generated a panel of LOV domain variants by mutating the key chromophore-proximal glutamine aminoacid of a thermostable flavin based fluorescent protein CagFbFP (Gln148) to asparagine, aspartate, glutamate, histidine, lysine and arginine. Absorption spectra of all of the mutants are blue-shifted, with the maximal shift of 8 nm observed for the Q148H variant. While CagFbFP and its Q148N/D/E variants are not sensitive to pH, Q148H/K/R reveal a moderate red shift induced byacidic pH. To gain further insight, we determined high resolution crystal structures of all of the mutants studied at the resolutions from 1.07 Å for Q148D to 1.63 Å for Q148R. Whereas in some of the variants, the aminoacid 148 remains in the vicinity of the flavin, in Q148K, Q148R and partially Q148D, the C-terminus of the protein unlatches and the side chain of the residue 148 is reoriented away from the chromophore. Our results explain the absence of color shifts from replacing Gln148 with charged aminoacids and pave the way for rational design of color-shifted flavin based fluorescent proteins.

The emergence of poly (ADP-ribose) polymerase (PARP) inhibitors targeting a class of PARP enzymes has gained a great interest in cancer therapy. Majority of the PARP inhibitors are not isoform-selective which may cause unwanted off-target effects. In the present study, we explore the molecular mechanism and energy requirements for PARP-2 inhibition. This involves docking studies, frontier molecular orbital analysis, 500 ns molecular dynamics simulation (MD), binding free energy analysis and principal component analysis. The results clearly suggest the importance of hydrogen bonding (Gly429, Gln332, Ser470, Tyr455) and π-π stacking interactions (His428, Tyr455, Tyr462, Phe463, Tyr473) between residues and the inhibitor. Presence of lowest unoccupied molecular orbitals favors π-π stacking interactions and highest occupied molecular orbital orbital favors hydrogen-bonding interactions in the ligands. The stability of most active/PARP-2 complex is confirmed by hydrogen bonding and π-π stacking interaction parameters. Molecular-mechanics Poisson-Boltzmann surface area energy calculations showed that van der Waals and nonpolar solvation energy terms are crucial components for the stable binding of the ligands. Per residue analysis showed that tyrosine, histidine, and phenyl alanine residues are responsible for hydrophobic interactions with the ligands. Four new inhibitors are designed based on this study and the stability of PARP-2/inhibitor complex is validated by MD, density functional theory studies, and ADME/toxicity properties. Information from the present study can serve as a basis for designing new isoform-selective PARP-2 inhibitors.

Nitrile hydratase (NHase), an excellent bio-catalyst for the synthesis of amide compounds, was composed of two heterologous subunits. A thermoalkaliphilic NHase NHCTA1 (Tm = 71.3°C) obtained by in silico screening in our study exhibited high flexibility of α-subunit but excellent thermostability, as opposed to previous examples. To gain a deeper structural insight into the thermostability of NHCTA1, comparative molecular dynamics simulation of NHCTA1 and reported NHases was carried out. By comparison, we speculated that β-subunit played a key role in adjusting the flexibility of α-subunit and the different conformations of linker in "α5-helix-coil ring" supersecondary structure of β-subunit can affect the interaction between β-subunit and α-subunit. Mutant NHCTA1-α6 C with a random coil linker and mutant NHCTA1-αβγ with a truncated linker were therefore constructed to understand the impact on NHCTA1 thermostability by varying the supersecondary structure. The varied thermostability of NHCTA1-α6 C and NHCTA1-αβγ (Tmα6C = 74.4°C, Tmαβγ = 65.6°C) verified that the flexibility of α-subunit adjusted by β-subunit was relevant to the stability of NHCTA1. This study gained an insight into the NNHCTA1 thermostability by virtual dynamics comparison and experimental studies without crystallization, and this approach could be applied to other industrial-important enzymes.

As a key cellular sensor, the TRPV1 channel undergoes a gating transition from a closed state to an open state in response to many physical and chemical stimuli. This transition is regulated by small-molecule ligands including lipids and various agonists/antagonists, but the underlying molecular mechanisms remain obscure. Thanks to recent revolution in cryo-electron microscopy, a growing list of new structures of TRPV1 and other TRPV channels have been solved in complex with various ligands including lipids. Toward elucidating how ligand binding correlates with TRPV1 gating, we have performed extensive molecular dynamics simulations (with cumulative time of 20 μs), starting from high-resolution structures of TRPV1 in both the closed and open states. By comparing between the open and closed state ensembles, we have identified state-dependent binding sites for small-molecule ligands in general and lipids in particular. We further use machine learning to predict top ligand-binding sites as important features to classify the closed vs open states. The predicted binding sites are thoroughly validated by matching homologous sites in all structures of TRPV channels bound to lipids and other ligands, and with previous functional/mutational studies of ligand binding in TRPV1. Taken together, this study has integrated rich structural, dynamic, and functional data to inform future design of small-molecular drugs targeting TRPV1.

Old world scorpions produce an abundance of toxins called α-NaTx, which interfere with the fast inactivation of voltage-gated sodium channels. Their selectivity to channels of mammals or insects depends on a part of toxin named the specificity module. We report here the spatial structure of a major and broadly active toxin MeuNaTxα-1 from the venom of Mesobuthus eupeus. Notably, its specificity module is markedly different from other α-NaTx with known 3D structure. Close inspection shows that its conformation is a result of an interplay between protein motifs such as the nest and niche, which eventually shape α-NaTx structural diversity.

Symmetric proteins are currently of interest as they allow creation of larger assemblies and facilitate the incorporation of metal ions in the larger complexes. Recently this was demonstrated by the biomineralization of the cadmium-chloride nanocrystal via the Pizza designer protein. However, the mechanism behind this formation remained unclear. Here, we set out to investigate the mechanism driving the formation of this nanocrystal via truncation, mutation, and circular permutations. In addition, the interaction of other biologically relevant metal ions with these symmetric proteins to form larger symmetric complexes was also studied. The formation of the initial nanocrystal is shown to originate from steric strain, where His 58 induces a different rotameric conformation on His 73, thereby distorting an otherwise perfect planar ring of alternating cadmium and chlorine ions, resulting in the smallest nanocrystal. Similar highly symmetric complexes were also observed for the other biological relevant metal ions. However, the flexibility of the coordinating histidine residues allows each metal ion to adopt its preferred geometry leading to either monomeric or dimeric β-propeller units, where the metal ions are located at the interface between both propeller units. These results demonstrate that symmetric proteins are not only interesting to generate larger assemblies, but are also the perfect scaffold to create more complex metal based assemblies. Such metal protein assemblies may then find applications in bionanotechnology or biocatalysis.

In vertebrates, the mineralocorticoid receptor (MR) is a steroid-activated nuclear receptor (NR) that plays essential roles in water-electrolyte balance and blood pressure homeostasis. It belongs to the group of oxo-steroidian NRs, together with the glucocorticoid (GR), progesterone (PR), and androgen (AR) receptors. Classically, these oxo-steroidian NRs homodimerize and bind to specific genomic sequences to activate gene expression. NRs are multi-domain proteins, and dimerization is mediated by both the DNA (DBD) and ligand binding domains (LBDs), with the latter thought to provide the largest dimerization interface. However, at the structural level, the dimerization of oxo-steroidian receptors LBDs has remained largely a matter of debate and, despite their sequence homology, there is currently no consensus on a common homodimer assembly across the four receptors, that is, GR, PR, AR, and MR. Here, we examined all available MR LBD crystals using different computational methods (protein common interface database, proteins, interfaces, structures and assemblies, protein-protein interaction prediction by structural matching, and evolutionary protein-protein interface classifier, and the molecular mechanics Poisson-Boltzmann surface area method). A consensus is reached by all methods and singles out an interface mediated by helices H9, H10 and the C-terminal F domain as having characteristics of a biologically relevant assembly. Interestingly, a similar assembly was previously identified for GRα, MR closest homolog. Alternative architectures that were proposed for GRα were not observed for MR. These data call for further experimental investigations of oxo-steroid dimer architectures.