Biophysics Seminar day - 9/16/2016 - 11:00am-1:30pm - 1080 Physics Research Building

11:00-12:00 Computational and Biophysical Studies of Unusual Cytochromes P450
John Hackett, Virginia Commonwealth University

The heme-thiolate cytochromes P450 (CYPs) are the most synthetically versatile enzymes, occupying an indispensable niche in hormone biosyntheses and in the disposition of xenobiotics. My laboratory has adopted the synergistic approach of integrating computational tools and experimental biophysical methods to elucidate structure-function relationships in those P450s that catalyze difficult or seemingly impossible transformations. This seminar will summarize our recent work on two enzymes, CYP19A1 (aromatase) and CYP4B1. The three-step conversion of androgens to estrogens by CYP19A1 is arguably the most mechanistically-enticing albeit controversial sequential P450 transformation. Results of hybrid quantum mechanics/molecular mechanics computational studies of the final CYP19A1 catalytic step as well those from experimental resonance Raman spectroscopy that support aspects of the proposed mechanism will be described. Members of the CYP4 family, including CYP4B1, are hydrocarbon ω-hydroxylases that preferentially activate terminal C-H bonds over their weaker congeners within the hydrocarbon chain. Spectroscopic evidence for a dramatic substrate-induced heme distortion will be presented and possible implications of this phenomenon on the regioselectivity of hydroxylation by this enzyme will be discussed.

12:30-1:00 Site Selection Limits Retroviral Integration
Nathan Jones, Fishel lab

Retroviruses must integrate their linear viral cDNA into the host genome for a productive infection. Integration is catalyzed by the retrovirus-encoded integrase (IN), which forms a tetramer complex with the viral cDNA long terminal repeat (LTR) ends termed an intasome. IN then removes two 3'-nucleotides from both LTR ends and catalyzes strand transfer of the recessed 3'-hydroxyls into the target DNA separated by 4-6 bp. Here we have used multiple single molecule imaging tools to determine how the Prototype Foamy Virus (PFV) retroviral intasome searches for an integration site. Once a target site is identified the PFV intasome catalyzes the two strand transfer events. We investigate the mechanism of those events. Further observations are made about possible DNA targets.

1:00-1:30 Structural variations in the cadherin-23 ectodomain and the molecular mechanisms of inherited deafness
Avinash Jaiganesh, Sotomayor lab

Inner ear hair cells convert the force stimulus of sound into an electrical signal through mechanotransduction. A core component of the mechanotransduction apparatus is the tip link, a protein filament that is essential for hearing. Each tip-link is made of two non-classical cadherins, cadherin-23 (cdh23) and protocadherin-15 (pcdh15), which interact through their N-terminal, extracellular cadherin [EC] repeats, in a calcium dependent manner. Each of these cadherins also forms parallel (cis) homodimers and the cdh23-pcdh15 complex is an anti-parallel (trans) heterotetramer. With 27 EC repeats, cdh23 constitutes 3/4th of the tip link. The linkers between EC repeats coordinate three calcium ions through highly conserved calcium-binding residues. These ions dictate EC repeat rigidity and are critical for cdh23 function. Over 100 missense mutations on cdh23 are known to be associated with deafness. These mutations either affect calcium binding, protein folding or possible cis-interaction interfaces. To examine the structural variations in the cdh23 ectodomain, its mechanical properties and the effects of deafness-related mutations, we obtained structures of 11 cdh23 EC repeats.

We used X-ray crystallography to solve the structures of wild-type (WT) cdh23 EC6-8 (2.89 Å), EC12-13 (2.59 Å), EC19-21 (2.78 Å) and EC22-24 (1.85 Å). These structures elucidate structural variations in otherwise similar EC repeats. Additionally, structures of EC19-21 fragments with deafness associated mutations D2148N (2.86 Å) and R2029W (2.85 Å) show how these mutations affect cdh23. We also used Differential Scanning Fluorimetry (DSF) to quantify decreased folding stability and calcium affinity due to other missense mutations in EC19-21. Molecular dynamics simulations are used to compare the mechanical strength and stiffness of different ectodomain fragments as well as to the disease related mutants. Overall, we present novel structures of cdh23 EC repeats and a comprehensive model for deafness due to missense mutations in cdh23.

Last update: 9/9/2016, Ralf Bundschuh