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1.  Structural basis for mu-opioid receptor binding and activation 
Structure (London, England : 1993)  2011;19(11):1683-1690.
Summary
Opioids that stimulate the μ-opioid receptor (MOR1) are the most frequently prescribed and effective analgesics. Here we present a structural model of MOR1. Molecular dynamics simulations show a ligand-dependent increase in the conformational flexibility of the third intracellular loop that couples with the G-protein complex. These simulations likewise identified residues that form frequent contacts with ligands. We validated the binding residues using site-directed mutagenesis coupled with radioligand binding and functional assays. The model was used to blindly screen a library of ~1.2 million compounds. From the thirty-four compounds predicted to be strong binders, the top three candidates were examined using biochemical assays. One compound showed high efficacy and potency. Post hoc testing revealed this compound to be nalmefene, a potent clinically used antagonist, thus further validating the model. In summary, the MOR1 model provides a tool for elucidating the structural mechanism of ligand-initiated cell signaling and screening for novel analgesics.
doi:10.1016/j.str.2011.08.003
PMCID: PMC3217204  PMID: 22078567
2.  Multiscale approaches for studying energy transduction in dynein 
Cytoplasmic dynein is an important motor that drives all minus-end directed movement along microtubules. Dynein is a complex motor whose processive motion is driven by ATP-hydrolysis. Dynein's run length has been measured to be several millimetres with typical velocities in the order of a few nanometres per second. Therefore, the average time between steps is a fraction of a second. When this time scale is compared with typical time scales for protein side chain and backbone movements (~10−9 s and ~10−5 s, respectively), it becomes clear that a multi-timescale modelling approach is required to understand energy transduction in this protein. Here, we review recent efforts to use computational and mathematical modelling to understand various aspects of dynein's chemomechanical cycle. First, we describe a structural model of dynein's motor unit showing a heptameric organization of the motor subunits. Second, we describe our molecular dynamics simulations of the motor unit that are used to investigate the dynamics of the various motor domains. Third, we present a kinetic model of the coordination between the two dynein heads. Lastly, we investigate the various potential geometries of the dimer during its hydrolytic and stepping cycle.
doi:10.1039/b902028d
PMCID: PMC2823375  PMID: 19506759
3.  A Structural Model of the Pore-Forming Region of the Skeletal Muscle Ryanodine Receptor (RyR1) 
PLoS Computational Biology  2009;5(4):e1000367.
Ryanodine receptors (RyRs) are ion channels that regulate muscle contraction by releasing calcium ions from intracellular stores into the cytoplasm. Mutations in skeletal muscle RyR (RyR1) give rise to congenital diseases such as central core disease. The absence of high-resolution structures of RyR1 has limited our understanding of channel function and disease mechanisms at the molecular level. Here, we report a structural model of the pore-forming region of RyR1. Molecular dynamics simulations show high ion binding to putative pore residues D4899, E4900, D4938, and D4945, which are experimentally known to be critical for channel conductance and selectivity. We also observe preferential localization of Ca2+ over K+ in the selectivity filter of RyR1. Simulations of RyR1-D4899Q mutant show a loss of preference to Ca2+ in the selectivity filter as seen experimentally. Electrophysiological experiments on a central core disease mutant, RyR1-G4898R, show constitutively open channels that conduct K+ but not Ca2+. Our simulations with G4898R likewise show a decrease in the preference of Ca2+ over K+ in the selectivity filter. Together, the computational and experimental results shed light on ion conductance and selectivity of RyR1 at an atomistic level.
Author Summary
Ryanodine receptors (RyRs) are ion channels present in the membranes of an intracellular calcium storage organelle, the sarcoplasmic reticulum. Nerve impulse triggers the opening of RyR channels, thus increasing the cytoplasmic calcium levels, which subsequently leads to muscle contraction. Congenital mutations in a specific type of RyR that is present in skeletal muscles, RyR1, lead to central core disease (CCD), which leads to weakened muscle. RyR1 mutations also render patients to be highly susceptible to malignant hyperthermia, an adverse reaction to general anesthesia. Although it is generally known that CCD mutations abort RyR1 function, the molecular basis of RyR1 dysfunction remains largely unknown because of the lack of atomic-level structure. Here, we present a structural model of the RyR1 pore region, where many of the CCD mutations are located. Molecular dynamics simulations of the pore region confirm the positions of residues experimentally known to be relevant for function. Furthermore, electrophysiological experiments and simulations shed light on the loss of function of CCD mutant channels. The combined theoretical and experimental studies on RyR1 elucidate the ion conduction pathway of RyR1 and a potential molecular origin of muscle diseases.
doi:10.1371/journal.pcbi.1000367
PMCID: PMC2668181  PMID: 19390614
4.  Identification and Rational Redesign of Peptide Ligands to CRIP1, A Novel Biomarker for Cancers 
PLoS Computational Biology  2008;4(8):e1000138.
Cysteine-rich intestinal protein 1 (CRIP1) has been identified as a novel marker for early detection of cancers. Here we report on the use of phage display in combination with molecular modeling to identify a high-affinity ligand for CRIP1. Panning experiments using a circularized C7C phage library yielded several consensus sequences with modest binding affinities to purified CRIP1. Two sequence motifs, A1 and B5, having the highest affinities for CRIP1, were chosen for further study. With peptide structure information and the NMR structure of CRIP1, the higher-affinity A1 peptide was computationally redesigned, yielding a novel peptide, A1M, whose affinity was predicted to be much improved. Synthesis of the peptide and saturation and competitive binding studies demonstrated approximately a 10–28-fold improvement in the affinity of A1M compared to that of either A1 or B5 peptide. These techniques have broad application to the design of novel ligand peptides.
Author Summary
Breast cancer is one of the most frequently diagnosed malignancies in American females and is the second leading cause of cancer deaths in women. Several improvements in diagnostic protocols have enhanced our ability for earlier detection of breast cancer, resulting in improvement of therapeutic outcome and an increased survival rate for breast cancer patients. However, current early screening techniques are neither comprehensive nor infallible. Imaging techniques that improve breast cancer detection, localization, and evaluation of therapy are essential in combating the disease. Cysteine-rich intestinal protein 1 (CRIP1) has been identified as a novel marker for early detection of breast cancers. Here, we report the use of phage display and computational molecular modeling to identify a high-affinity ligand for CRIP1. Phage display panning experiments initially identified consensus peptide sequences with modest binding affinity to purified CRIP1. Using ab initio modeling of binding peptide structures, computational docking, and recently developed free energy estimation protocols, we redesigned the peptides to increase their affinity for CRIP1. Synthesis of the redesigned peptide and binding studies demonstrated approximately a 10–28-fold improvement in the binding affinity. The combination of computational and experimental techniques in this study demonstrates a potentially powerful tool in modulating protein–protein interactions.
doi:10.1371/journal.pcbi.1000138
PMCID: PMC2453235  PMID: 18670594
5.  Diminished Self-Chaperoning Activity of the ΔF508 Mutant of CFTR Results in Protein Misfolding 
PLoS Computational Biology  2008;4(2):e1000008.
The absence of a functional ATP Binding Cassette (ABC) protein called the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) from apical membranes of epithelial cells is responsible for cystic fibrosis (CF). Over 90% of CF patients carry at least one mutant allele with deletion of phenylalanine at position 508 located in the N-terminal nucleotide binding domain (NBD1). Biochemical and cell biological studies show that the ΔF508 mutant exhibits inefficient biosynthetic maturation and susceptibility to degradation probably due to misfolding of NBD1 and the resultant misassembly of other domains. However, little is known about the direct effect of the Phe508 deletion on the NBD1 folding, which is essential for rational design strategies of cystic fibrosis treatment. Here we show that the deletion of Phe508 alters the folding dynamics and kinetics of NBD1, thus possibly affecting the assembly of the complete CFTR. Using molecular dynamics simulations, we find that meta-stable intermediate states appearing on wild type and mutant folding pathways are populated differently and that their kinetic accessibilities are distinct. The structural basis of the increased misfolding propensity of the ΔF508 NBD1 mutant is the perturbation of interactions in residue pairs Q493/P574 and F575/F578 found in loop S7-H6. As a proof-of-principle that the S7-H6 loop conformation can modulate the folding kinetics of NBD1, we virtually design rescue mutations in the identified critical interactions to force the S7-H6 loop into the wild type conformation. Two redesigned NBD1-ΔF508 variants exhibited significantly higher folding probabilities than the original NBD1-ΔF508, thereby partially rescuing folding ability of the NBD1-ΔF508 mutant. We propose that these observed defects in folding kinetics of mutant NBD1 may also be modulated by structures separate from the 508 site. The identified structural determinants of increased misfolding propensity of NBD1-ΔF508 are essential information in correcting this pathogenic mutant.
Author Summary
Deletion of a single residue, phenylalanine at position 508, in the first nucleotide binding domain (NBD1) of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is present in approximately 90% of cystic fibrosis (CF) patients. Experiments show that this mutant protein exhibits inefficient biosynthetic maturation and susceptibility to degradation probably due to misfolding of NBD1 and the resultant incorrect interactions of other domains. However, little is known about the direct effect of the Phe508 deletion on NBD1 folding. Here, using molecular dynamics simulations of NBD1-WT, NBD1-F508A, and NBD1-ΔF508, we show that the deletion of Phe508 indeed alters the kinetics of NBD1 folding. We also find that the intermediate states appearing on wild type and mutant folding pathways are populated differently and that their kinetic accessibilities are distinct. Moreover, we identified critical interactions not necessarily localized near position 508, such as Q493/P574 and F575/F587, to be significant structural elements influencing the kinetic difference between wild type and mutant NBD1. We propose that these observed alterations in folding kinetics of mutant NBD1 result in misassembly of the whole multi-domain protein, thereby causing its premature degradation.
doi:10.1371/journal.pcbi.1000008
PMCID: PMC2265529  PMID: 18463704

Results 1-5 (5)