Structure-based drug design is frequently used to accelerate the development of small-molecule therapeutics. Although substantial progress has been made in X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy, the availability of high-resolution structures is limited owing to the frequent inability to crystallize or obtain sufficient NMR restraints for large or flexible proteins. Computational methods can be used to both predict unknown protein structures and model ligand interactions when experimental data are unavailable. This paper describes a comprehensive and detailed protocol using the Rosetta modeling suite to dock small-molecule ligands into comparative models. In the protocol presented here, we review the comparative modeling process, including sequence alignment, threading and loop building. Next, we cover docking a small-molecule ligand into the protein comparative model. In addition, we discuss criteria that can improve ligand docking into comparative models. Finally, and importantly, we Reprints and permissions information is available online at http://www.nature.com/reprints/index.html.Correspondence should be addressed to J.M. (jens.meiler@vanderbilt.edu). 7 These authors contributed equally to this work.Note: Supplementary information is available in the online version of the paper. AUTHOR CONTRIBUTIONSAll authors contributed equally to this work. All authors wrote substantial portions of the main text, the figures and the supplementary information. S.A.C. proposed the composition of the work for the benefit of the scientific community, tested the presented protocol and managed submission. S.L.D. wrote instructions on how to install the software, generated the comparative models, wrote dataprocessing scripts and managed references. S.H.D. wrote the supplementary glossary and was responsible for overall editing of the work. G.H.L. wrote the RosettaLigand program in its present form. D.P.N. carefully read through the manuscript for consistency and accuracy and helped in the analysis of the generated models. E.D.N. also generated comparative models, performed all of the ligand docking and performed the data analysis. J.R.W. contributed several figures, data-processing scripts, specialty movers, wrote large sections of the tutorial and managed references. J.H.S. tested the protocol, wrote the Troubleshooting section and edited the manuscript for clarity. J.M. helped define the scope of the work and guided the process of establishing the protocol. COMPETING FINANCIAL INTERESTSThe authors declare no competing financial interests. Author Manuscript present a strategy for assessing model quality. The entire protocol is presented on a single example selected solely for didactic purposes. The results are therefore not representative and do not replace benchmarks published elsewhere. We also provide an additional tutorial so that the user can gain hands-on experience in using Rosetta. The protocol should take 5-7 h, with additional time allocated for computer generation of models....

KCNE3 modulates the KCNQ1 K+ channel in epithelia by directly stabilizing the voltage-sensor S4 segment in its activated state.

Bacterial phosphopentomutases (PPMs) are alkaline phosphatase superfamily members that interconvert ␣-D-ribose 5-phosphate (ribose 5-phosphate) and ␣-D-ribose 1-phosphate (ribose 1-phosphate). We investigated the reaction mechanism of Bacillus cereus PPM using a combination of structural and biochemical studies. Enzyme-catalyzed phosphoryl transfer forms the basis for many biological, bioenergetic, and regulatory processes and is one of the most common cellular reactions (1). Numerous enzyme families have evolved mechanistically distinct solutions for phosphoryl transfer (2). Phosphomutases are phosphotransfer enzymes that rearrange the position of phosphate within a substrate molecule through either intramolecular (i.e. the phosphate is transferred to a different position on the same molecule) or intermolecular phosphoryl transfer (i.e. the phosphate is transferred from one substrate molecule to another).Bacterial phosphopentomutases (PPMs) 3 (EC 5.4.2.7) interconvert ribose 1-phosphate and ribose 5-phosphate, which bridges glucose metabolism and RNA biosynthesis (3). The importance of this reaction has recently been underscored by the observation that targeted deletion of the gene encoding PPM in the pathogen Francisella tularensis (deoB) results in markedly decreased virulence (4). PPMs appear to be biochemically and structurally distinct from their human congeners (5, 6), making them potential targets for antibiotic development.Sequence clustering classifies prokaryotic PPMs within the alkaline phosphatase superfamily of metalloenzymes, which includes a range of functionally diverse enzymes such as cofactor-independent phosphoglycerate mutase, phosphodiesterase, and estrone and aryl sulfatases (7). The majority of alkaline phosphatase superfamily enzymes catalyze a hydrolase reaction; however, both PPM (5) and the cofactor-independent phosphoglycerate mutase catalyze phosphomutase reactions (8, 9).All previously characterized alkaline phosphatase superfamily members follow a unified general reaction mechanism ( Fig. 1) (10). In alkaline phosphatase itself (11, 12), the catalytically competent enzyme has an unphosphorylated catalytic nucleophile, Ser-102 (Fig. 1, state 1). Turnover is initiated when the metallocenter activates a phosphoester donor substrate (R D -OPO 3 H Ϫ ) (Fig. 1, state 2) to transfer the phosphoryl group to the hydroxyl of Ser-102 (Fig. 1, state 3). This results in a covalent phosphoenzyme intermediate (E-OPO 3 H Ϫ ) (Fig. 1, state 4). A second phosphoryl transfer from the enzyme to the acceptor water molecule (Fig. 1, states 5 and 6) completes the reaction cycle. This general reaction mechanism has also been verified * This work was supported, in whole or in part, by National Institutes of Health Grants GM079419 (to T. M. I.), GM077189 (to B. O. B.), GM051366 (to B. E. W.), DK070787 (to B. E. W.), T32 NS07491 (to T. D. P.), T32 GM008320 (to T. D. P.), T90 DA022873 (to D. P. N.), and T32 GM07628 (to G. R. W.). This work was also supported by a pilot award funded by the Vanderbilt Instit...

The FNT (formate-nitrite transporters) form a superfamily of pentameric membrane channels that translocate monovalent anions across biological membranes. FocA (formate channel A) translocates formate bidirectionally but the mechanism underlying how translocation of formate is controlled and what governs substrate specificity remains unclear. Here we demonstrate that the normally soluble dimeric enzyme pyruvate formate-lyase (PflB), which is responsible for intracellular formate generation in enterobacteria and other microbes, interacts specifically with FocA. Association of PflB with the cytoplasmic membrane was shown to be FocA dependent and purified, Strep-tagged FocA specifically retrieved PflB from Escherichia coli crude extracts. Using a bacterial two-hybrid system, it could be shown that the N-terminus of FocA and the central domain of PflB were involved in the interaction. This finding was confirmed by chemical cross-linking experiments. Using constraints imposed by the amino acid residues identified in the cross-linking study, we provide for the first time a model for the FocA–PflB complex. The model suggests that the N-terminus of FocA is important for interaction with PflB. An in vivo assay developed to monitor changes in formate levels in the cytoplasm revealed the importance of the interaction with PflB for optimal translocation of formate by FocA. This system represents a paradigm for the control of activity of FNT channel proteins.

Several live attenuated rotavirus (RV) vaccines have been licensed, but the mechanisms of protective immunity are still poorly understood. The most frequent human B cell response is directed to the internal protein VP6 on the surface of double-layered particles, which is normally exposed only in the intracellular environment. Here, we show that the canonical VP6 antibodies secreted by humans bind to such particles and inhibit viral transcription. Polymeric IgA RV antibodies mediated an inhibitory effect against virus replication inside cells during IgA transcytosis. We defined the recognition site on VP6 as a quaternary epitope containing a high density of charged residues. RV human mAbs appear to bind to a negatively-charged patch on the surface of the Type I channel in the transcriptionally active particle, and they sterically block the channel. This unique mucosal mechanism of viral neutralization, which is not apparent from conventional immunoassays, may contribute significantly to human immunity to RV.