University, New York, NY, USA. Proteins carrying out the ... Chemistry and Biochemistry, University of Minnesota Duluth, Duluth, MN,. USA. The primary role of ...
362a
Tuesday, March 1, 2016
high-resolution crystal structure of the drug-entrapped intermediate and exploited atomistic molecular dynamics simulations of the very large systems for up to a microsecond to investigate the dynamics of this process [1]. We observed a nearly vectorial transition of the de-poisoned complex toward the resealing-compliant configuration, and the statistical analysis revealed the non-concerted maneuvers of the two different DNA strands mediated by the enzyme. Using a neighborhood-based metric, we captured the important dynamical transitions during the re-ligation from the vast amount of correlated motions and provided the visible links between previously unsettled experimental observations. With the simulations of the drug-bound complexes, we also presented the conformational population shifts of an important residue in response to the binding of different drug molecules [2]. Furthermore, by comparing the coordinated dynamics revealed by different isoforms, we are able to dissect the differential dynamics of the critical DNA re-ligation conducted by the enzymes. We believe these are useful insights for the development of new anticancer drugs with lower cardiotoxicity. References: [1] Nucleic Acids Res. 2015, 43:6772-6786. [2] Molecules. 2014, 19:7415-7428. 1785-Plat From Physics to Phenotype: New Insights into Allosteric Transport Mechanisms in LeuT Michael V. LeVine, Michel A. Cuendet, George Khelashvili, Harel Weinstein. Physiology and Biophysics, Weill Cornell Medical College of Cornell University, New York, NY, USA. Proteins carrying out the many biological processes essential to cellular function act as biomolecular machines utilizing mechanisms that are most often allosteric in nature, i.e. the structural components involved in the mechanism, which are sometimes separated by large distances at the molecular scale, are coupled energetically. Although it is a ubiquitous mechanism, allostery is still poorly understood as an experimentally validated molecular mechanism underlying particular instances, and lacks a general theoretical approach to describe it quantitatively. We will present our efforts to address both of these inadequacies using the secondary active transporter LeuT as an example. First, we will introduce a model for secondary active transport (LeVine, Cuendet, Khelashvili, and Weinstein. In review) that implicitly includes the allosteric coupling between two separate processes: i) sodium and substrate binding, and ii) substrate binding and intracellular opening of the transporter to allow release. Next, a general theoretical approach for allostery will be addressed with a statistical mechanical model based on the Ising Model (LeVine and Weinstein, Entropy, 2015). This will be shown to provide analytical insight into how long-distance allosteric couplings, such as that occurring between substrate binding and intracellular opening in LeuT, can arise in a complex system of interacting structural components. Finally, we will address the lack of experimentally validated molecular mechanisms underlying allostery. We will discuss a proposed model for substrate binding/intracellular opening coupling in LeuT, discovered using a novel information theory-based analysis framework, N-body Information Theory (NbIT) (LeVine and Weinstein, PLoS Comp Biol, 2014), and will present a recent experimental validation of this model. This presentation of combined theoretical, computational, and experimental work will demonstrate how deeper mechanistic understanding of biomolecular machines can be achieved through improved models of allosteric behavior. 1786-Plat Pleiotropic Role Played by the PDZ Domain in Neuronal Signaling Pathways Ce´lia Caillet-Saguy1, Pierre Maisonneuve1, Florent Delhommel1, Henri Buc2, Monique Lafon3, Muriel Delepierre1, Florence Cordier1, Nicolas Wolff1. 1 Structural Biology and Chemistry, Institut Pasteur, Paris, France, 2Institut Pasteur, Paris, France, 3Virology, Institut Pasteur, Paris, France. The human tyrosine phosphatase PTPN4 and the Ser/Thr kinase MAST2 are two enzymes expressed in neurons. While PTPN4 is an anti-apoptotic protein, MAST2 inhibits neurogenesis and neuroprotection. The PDZ domain of these two enzymes is specifically targeted by the glycoprotein of the rabies virus during neuron infection. We have solved the NMR and X-ray structures of the complexes formed by MAST2-PDZ and PTPN4-PDZ with their respectives endogenous and viral ligands. As a result, the complexes formed by the PDZ of the two enzymes and their respective ligands are disrupted, triggering drastic effect on cell signaling and cell commitment either towards death or survival. The glycoprotein disrupts the interactions of MAST2 and PTPN4 PDZ domains with their respective cellular ligands, the phosphatase PTEN and the MAP kinase p38g.
The PDZ domains of MAST2 and PTPN4 contribute to the recruitment of substrates but also to the catalytic regulation modulating the phosphorylation/ dephosphorylation of endogenous partners. On the one hand, the binding of PTEN to the PDZ domain of MAST2 prevents MAST2 auto-association and drastically increases the phosphorylation level of PTEN. We identified by NMR two independent cascades of PTEN phosphorylation, in vitro and in cell extracts, that could activate different regulatory responses of the phosphatase. On the other hand, we combine X-ray crystallography, SAXS and NMR, to show that the PDZ domain of PTPN4 inhibits the catalytic activity of the flanking phosphatase domain and the mere binding of the p38g PDZ binding sequence is sufficient to allosterically restore the catalytic competence of PTPN4 by disrupting the inter-domain communication. Caillet-Saguy et al. (2015) Prog Biophys Mol Biol. 119(1):53-9. Delhommel F et al. (2015) Biochemical Journal 469(1) :159-168. Vincentelli R et al. (2015) Nature Methods 8 :787-793. Cordier, F.,et al.(2015) Methods, 77-78:82-91. 1787-Plat Hydrophobic Interactions Elicit Cooperative Response in Dystrophin Alessandro Cembran, Anne Hinderliter, Benjamin T. Horn, Caitlin T. Pederson, Katie L. Schneider, Jesse A. Skogstad. Chemistry and Biochemistry, University of Minnesota Duluth, Duluth, MN, USA. The primary role of dystrophin, a 427 kDa elongated protein, is to stabilize the membrane of muscle cells against the mechanical forces deriving from muscle contraction and relaxation; its absence or mutation lead to various forms of muscular dystrophy. Dystrophin binds to actin through its N-terminus actin binding domain (ABD1), and connects to the cell membrane through its C-terminus; in between, 24 spectrin repeats (SR) with interspersed hinges provide structural flexibility and have been proposed to actively dissipate the mechanical stress. Yet, dystrophin’s mechanism of function is largely unknown, preventing effective drug development. Our working hypothesis is that dystrophin’s domains are allosterically coupled through specific hydrophobic interactions, and that the response to mechanical stress is propagated across domains through order-to-disorder transitions at their interface. We test this hypothesis with all-atom molecular dynamics simulations of dystrophin’s SR 1 monomer, SR 17-18 dimer, and the ABD1 domain. Our results support the hypothesis by showing that (a) forced unfolding by pulling initiates at the interface between SR repeats; (b) the linker between SR 17-18 is flexible and allows for distinct conformations at the interface between domains, characterized by different hydrophobic interaction patterns; (c) mutual information analyses show that conformational changes at the dimer’s interface and termini are coupled; and (d) hydrophobic interactions between the two CH domains and the connecting linker stabilize a closed conformation of ABD1 that is alternative to the open crystal structure and explains experimental fluorescence results. Our results suggest that the disease may originate from altered hydrophobic interactions, and pave the way for targeted drug development.
Platform: RNA Structure and Dynamics 1788-Plat Interrogation of CRISPR Dynamics with Fluorescent Single Guide RNAs in Live Cells Hanhui Ma1, Li-Chun Tu2, Ardalan Naseri3, Shaojie Zhang3, Maximilliaan Huisman2, David Grunwald2, Thoru Pederson1. 1 Department of Biochemistry and Molecular Pharmacology, UMASS Medical School, Worcester, MA, USA, 2RNA Therapeutics Institute, UMASS Medical School, Worcester, MA, USA, 3Department of Electrical Engineering and Computer Science, University of Central Florida, Orlando, FL, USA. Although the CRISPR-Cas9 system has been widely used for genome editing, little is known about its dynamics in live cells such as the kinetics of sgRNA:Cas9 assembly and DNA recognition. By stably expressing both fluorescent dCas9 and sgRNAs that specifically targets on chromosome 3 in live human cells we monitored the assembly of sgRNA:dCas9 complexes and tracked its dynamics of interactions with the target gene. In the absence of dCas9 expression, the sgRNA was so unstable as to not be detectable. In the presence of dCas9 the sgRNA was primarily localized in the nucleus and displayed a half-life within 15 minutes estimated by actinomycin chase experiments. These results are in agreement with sgRNA stability and sgRNA:dCas9 complex formation being the limiting step for DNA targeting. We next monitored the dynamics of on-target dCas9:sgRNA complexes by fluorescence recovery after photobleaching (FRAP), employing different lengths of sgRNA seed sequence
