We describe herein a computationally intensive project aimed at carrying out molecular dynamics (MD) simulations including water and counterions on B-DNA oligomers containing all 136 unique tetranucleotide base sequences. This initiative was undertaken by an international collaborative effort involving nine research groups, the "Ascona B-DNA Consortium" (ABC). Calculations were carried out on the 136 cases imbedded in 39 DNA oligomers with repeating tetranucleotide sequences, capped on both ends by GC pairs and each having a total length of 15 nucleotide pairs. All MD simulations were carried out using a well-defined protocol, the AMBER suite of programs, and the parm94 force field. Phase I of the ABC project involves a total of approximately 0.6 mus of simulation for systems containing approximately 24,000 atoms. The resulting trajectories involve 600,000 coordinate sets and represent approximately 400 gigabytes of data. In this article, the research design, details of the simulation protocol, informatics issues, and the organization of the results into a web-accessible database are described. Preliminary results from 15-ns MD trajectories are presented for the d(CpG) step in its 10 unique sequence contexts, and issues of stability and convergence, the extent of quasiergodic problems, and the possibility of long-lived conformational substates are discussed.

Newly generated actin free barbed ends at the front of motile cells provide sites for actin filament assembly driving membrane protrusion. Growth factors induce a rapid biphasic increase in actin free barbed ends, and we found both phases absent in fibroblasts lacking H+ efflux by the Na-H exchanger NHE1. The first phase is restored by expression of mutant cofilin-H133A but not unphosphorylated cofilin-S3A. Constant pH molecular dynamics simulations and nuclear magnetic resonance (NMR) reveal pH-sensitive structural changes in the cofilin C-terminal filamentous actin binding site dependent on His133. However, cofilin-H133A retains pH-sensitive changes in NMR spectra and severing activity in vitro, which suggests that it has a more complex behavior in cells. Cofilin activity is inhibited by phosphoinositide binding, and we found that phosphoinositide binding is pH-dependent for wild-type cofilin, with decreased binding at a higher pH. In contrast, phosphoinositide binding by cofilin-H133A is attenuated and pH insensitive. These data suggest a molecular mechanism whereby cofilin acts as a pH sensor to mediate a pH-dependent actin filament dynamics.

Actin filament binding by the focal adhesion (FA)-associated protein talin stabilizes cell-substrate adhesions and is thought to be rate-limiting in cell migration. Although F-actin binding by talin is known to be pH-sensitive in vitro, with lower affinity at higher pH, the functional significance of this pH dependence is unknown. Because increased intracellular pH (pHi) promotes cell migration and is a hallmark of metastatic carcinomas, we asked whether it increases FA remodeling through lower-affinity talin-actin binding. Talin contains several actin binding sites, but we found that only the COOH-terminal USH-I/LWEQ module showed pH-dependent actin binding, with lower affinity and decreased maximal binding at higher pH. Molecular dynamics simulations and NMR of this module revealed a structural mechanism for pH-dependent actin binding. A cluster of titratable amino acids with upshifted pKa values, including His-2418, was identified at one end of the five-helix bundle distal from the actin binding site. Protonation of His-2418 induces changes in the conformation and dynamics of the remote actin binding site. Structural analyses of a mutant talin-H2418F at pH 6.0 and 8.0 suggested changes different from the WT protein, and we confirmed that actin binding by talin-H2418F was relatively pH-insensitive. In motile fibroblasts, increasing pHi decreased FA lifetime and increased the migratory rate. However, expression of talin-H2418F increased lifetime 2-fold and decreased the migratory rate. These data identify a molecular mechanism for pH-sensitive actin binding by talin and suggest that FA turnover is pH-dependent and in part mediated by pH-dependent affinity of talin for binding actin. intracellular pH ͉ NHE1 ͉ migration F ocal adhesion (FA) remodeling is a rate-limiting determinant in haptokinetic migration of adherent cells. At the leading edge of migrating cells, FAs undergo rapid cycles of assembly and turnover, creating and disrupting, respectively, sites of traction necessary for forward movement of the cell body. Force generation for traction requires linkage among the extracellular matrix, integrin receptors, and actin filaments. Actin filaments do not directly bind to the cytoplasmic domain of integrins but bind to integrin-associated FA proteins such as talin and vinculin. Although several mechanisms contribute to FA remodeling in migrating cells (1), emerging evidence indicates that talin plays a central role in the dynamic linkage between integrins and actin filaments necessary for cell migration (2, 3). Talin functions in distinct albeit complementary mechanisms that promote FA turnover. First is cleavage of talin by the protease calpain, which also modulates adhesion complex composition and likely signaling functions of talin (4). Second is regulated talin binding to actin filaments, which is proposed to act as a clutch to control FA turnover and membrane protrusion dynamics (3, 5-7). How actin binding by talin is dynamically regulated during cell migration, however, remains undetermined.Previous stu...

Dihydrofolate reductase (DHFR) is the enzyme responsible for the NADPH-dependent reduction of 5,6-dihydrofolate to 5,6,7,8-tetrahydrofolate, an essential cofactor in the synthesis of purines, thymidylate, methionine, and other key metabolites. Because of its importance in multiple cellular functions, DHFR has been the subject of much research targeting the enzyme with anticancer, antibacterial, and antimicrobial agents. Clinically used compounds targeting DHFR include methotrexate for the treatment of cancer and diaminopyrimidines (DAPs) such as trimethoprim (TMP) for the treatment of bacterial infections. DAP inhibitors of DHFR have been used clinically for >30 years and resistance to these agents has become widespread. Methicillin-resistant Staphylococcus aureus (MRSA), the causative agent of many serious nosocomial and community acquired infections, and other gram-positive organisms can show resistance to DAPs through mutation of the chromosomal gene or acquisition of an alternative DHFR termed ''S1 DHFR.'' To develop new therapies for health threats such as MRSA, it is important to understand the molecular basis of DAP resistance. Here, we report the crystal structure of the wild-type chromosomal DHFR from S. aureus in complex with NADPH and TMP. We have also solved the structure of the exogenous, TMP resistant S1 DHFR, apo and in complex with TMP. The structural and thermodynamic data point to important molecular differences between the two enzymes that lead to dramatically reduced affinity of DAPs to S1 DHFR. These differences in enzyme binding affinity translate into reduced antibacterial activity against strains of S. aureus that express S1 DHFR. Proteins 2009; 76:706-717. V V C 2009 Wiley-Liss, Inc.

We present a thermodynamical approach to identify changes in macromolecular structure and dynamics in response to perturbations such as mutations or ligand binding, using an expansion of the Kullback-Leibler Divergence that connects local population shifts in torsion angles to changes in the free energy landscape of the protein. While the Kullback-Leibler Divergence is a known formula from information theory, the novelty and power of our implementation lies in its formal developments, connection to thermodynamics, statistical filtering, ease of visualization of results, and extendability by adding higher-order terms. We present a formal derivation of the Kullback-Leibler Divergence expansion and then apply our method at a first-order approximation to molecular dynamics simulations of four protein systems where ligand binding or pH titration is known to cause an effect at a distant site. Our results qualitatively agree with experimental measurements of local changes in structure or dynamics, such as NMR chemical shift perturbations and hydrogen-deuterium exchange mass spectrometry. The approach produces easy-to-analyze results with low background, and as such has the potential to become a routine analysis when molecular dynamics simulations in two or more conditions are available. Our method is implemented in the MutInf code package and is available on the SimTK website at https://simtk.org/home/mutinf.