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The protein 5′-methylthioadenosine/ S-adenosylhomocysteine nucleosidase (MTAN) is involved in the quorum sensing of several bacterial species, including Helicobacter pylori. In particular, these bacteria depend on MTAN for synthesis of vitamin K homologs. The residue D198 in the active site of MTAN seems to be of crucial importance, by acting as a hydrogen-bond acceptor for the ligand. In this study, we investigated the conformation and dynamics of apo and holo H. pylori MTAN ( HpMTAN), and assessed the effect of protonation of D198 by use of molecular dynamics simulations. Our results show that protonation of the active site of HpMTAN can cause a conformational transition from a closed state to an open state even in the absence of substrate, via inter-chain mechanical coupling. [ABSTRACT FROM AUTHOR]

Hemoglobin (Hb), an oxygen-binding protein composed of four subunits (α1, α2, β1, and β2), is a well-known example of allosteric proteins that are capable of cooperative ligand binding. Despite decades of studies, the structural basis of its cooperativity remains controversial. In this study, we have integrated coarse-grained (CG) modeling, all-atom simulation, and structural data from X-ray crystallography and wide-angle X-ray scattering (WAXS), aiming to probe dynamic properties of the two structural states of Hb (T and R state) and the transitions between them. First, by analyzing the WAXS data of unliganded and liganded Hb, we have found that the structural ensemble of T or R state is dominated by one crystal structure of Hb with small contributions from other crystal structures of Hb. Second, we have used normal mode analysis to identify two distinct quaternary rotations between the α1β1 and α2β2 dimer, which drive the transitions between T and R state. We have also identified the hot-spot residues whose mutations are predicted to greatly change these quaternary motions. Third, we have generated a CG transition pathway between T and R state, which predicts a clear order of quaternary and tertiary changes involving α and β subunits in Hb. Fourth, we have used the accelerated molecular dynamics to perform an all-atom simulation starting from the T state of Hb, and we have observed a transition toward the R state of Hb. Further analysis of crystal structural data and the all-atom simulation trajectory has corroborated the order of quaternary and tertiary changes predicted by CG modeling. Proteins 2013. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Eukaryotic DNA polymerase (Pol) X family members such as Pol µ and terminal deoxynucleotidyl transferase (TdT) are important components for the nonhomologous DNA end-joining (NHEJ) pathway. TdT participates in a specialized version of NHEJ, V(D)J recombination. It has primarily nontemplated polymerase activity but can take instructions across strands from the downstream dsDNA, and both activities are highly dependent on a structural element called Loop1. However, it is unclear whether Pol µ follows the same mechanism, because the structure of its Loop1 is disordered in available structures. Here, we used a chimeric TdT harboring Loop1 of Polµ that recapitulated the functional properties of Pol µ in ligation experiments. We solved three crystal structures of this TdT chimera bound to several DNA substrates at 1.96-2.55 Å resolutions, including a full DNA double-strand break (DSB) synapsis. We then modeled the full Polµ sequence in the context of one these complexes. The atomic structure of an NHEJ junction with a Pol X construct that mimics Pol µ in a reconstituted system explained the distinctive properties of Pol µ compared with TdT. The structure suggested a mechanism of base selection relying on Loop1 and taking instructions via the in trans templating base independently of the primer strand. We conclude that our atomic-level structural observations represent a paradigm shift for the mechanism of base selection in the Pol X family of DNA polymerases. [ABSTRACT FROM AUTHOR]

The calcium-binding protein S100A4 plays an important role in a wide range of biological processes such as cell motility, invasion, angiogenesis, survival, differentiation, contractility, and tumor metastasis and interacts with a range of partners. To understand the functional roles and interplay of S100A4 binding partners such as Ca 2+ and nonmuscle myosin IIA (NMIIA), we used molecular dynamics simulations to investigate apo S100A4 and four holo S100A4 structures: S100A4 bound to Ca 2+ , S100A4 bound to NMIIA, S100A4 bound to Ca 2+ and NMIIA, and a mutated S100A4 bound to Ca 2+ and NMIIA. Our results show that two competing factors, namely, Ca 2+ -induced activation and NMIIA-induced inhibition, modulate the dynamics of S100A4 in a competitive manner. Moreover, Ca 2+ binding results in enhanced dynamics, regulating the interactions of S100A4 with NMIIA, while NMIIA induces asymmetric dynamics between the chains of S100A4. The results also show that in the absence of Ca 2+ the S100A4-NMIIA interaction is weak compared to that of between S100A4 bound to Ca 2+ and NMIIA, which may offer a quick response to dropping calcium levels. In addition, certain mutations are shown to play a marked role on the dynamics of S100A4. The results described here contribute to understanding the interactions of S100A4 with NMIIA and the functional roles of Ca 2+ , NMIIA, and certain mutations on the dynamics of S100A4. The results of this study could be interesting for the development of inhibitors that exploit the shift of balance between the competing roles of Ca 2+ and NMIIA.

Archaeal DNA polymerases from the B-family (polB) have found essential applications in biotechnology. In addition, some of their variants can accept a wide range of modified nucleotides or xenobiotic nucleotides, such as 1,5-anhydrohexitol nucleic acid (HNA), which has the unique ability to selectively cross-pair with DNA and RNA. This capacity is essential to allow the transmission of information between different chemistries of nucleic acid molecules. Variants of the archaeal polymerase from Thermococcus gorgonarius, TgoT, that can either generate HNA from DNA (TgoT_6G12) or DNA from HNA (TgoT_RT521) have been previously identified. To understand how DNA and HNA are recognized and selected by these two laboratory-evolved polymerases, we report six X-ray structures of these variants, as well as an in silico model of a ternary complex with HNA. Structural comparisons of the apo form of TgoT_6G12 together with its binary and ternary complexes with a DNA duplex highlight an ensemble of interactions and conformational changes required to promote DNA or HNA synthesis. MD simulations of the ternary complex suggest that the HNA-DNA hybrid duplex remains stable in the A-DNA helical form and help explain the presence of mutations in regions that would normally not be in contact with the DNA if it were not in the A-helical form. One complex with two incorporated HNA nucleotides is surprisingly found in a one nucleotide-backtracked form, which is new for a DNA polymerase. This information can be used for engineering a new generation of more efficient HNA polymerase variants.

In this chapter, we demonstrate the usage of a coarse-grained elastic network model to analyze protein conformational transitions in the NS3 helicase (NS3hel) of Hepatitis C virus (HCV). This analysis allows us to identify and visualize collective domain motions involved in the conformational transitions and predict the order of structural events during the transitions. It is highly efficient and applicable to many multi-domain protein structures which undergo large conformational changes to fulfill their functions. This method is made available through a Web server ( http://enm.lobos.nih.gov ).

To circumvent the difficulty of directly solving high-resolution biomolecular structures, low-resolution structural data from Cryo-electron microscopy (EM) and small angle solution X-ray scattering (SAXS) are increasingly used to explore multiple conformational states of biomolecular assemblies. One promising venue to obtain high-resolution structural models from low-resolution data is via data-constrained flexible fitting. To this end, we have developed a new method based on a coarse-grained Cα-only protein representation, and a modified form of the elastic network model (ENM) that allows large-scale conformational changes while maintaining the integrity of local structures including pseudo-bonds and secondary structures. Our method minimizes a pseudo-energy which linearly combines various terms of the modified ENM energy with an EM/SAXS-fitting score and a collision energy that penalizes steric collisions. Unlike some previous flexible fitting efforts using the lowest few normal modes, our method effectively utilizes all normal modes so that both global and local structural changes can be fully modeled with accuracy. This method is also highly efficient in computing time. We have demonstrated our method using adenylate kinase as a test case which undergoes a large open-to-close conformational change. The EM-fitting method is available at a web server (http://enm.lobos.nih.gov), and the SAXS-fitting method is available as a pre-compiled executable upon request., (© 2014 Elsevier Inc. All rights reserved.)