Simbios Talk by Andres Bayani-Tellez, Stanford University, Feb 28, 2006

Title: "Computational Modelling of Protein Oligomers."

This talk will be about computational modelling of protein oligomers- the basic idea is that one is given a pdb structure of a single protein or dimer and an EM image of some higher order structure, and the work is involved with fitting the crystal structure into the low resolution EM images. Modelling considerations include protein conformational changes, docking (geometric and energetic exploration of possible fits), clustering using spatial properties, bolstering the structual approach with conservation/covariation of a Multiple Sequence Alignment, as well as visualization and experimental validation. A case study (poliovirus RNA dependent RNA polymerase) will be discussed as well as early applications to FtsZ, the bacterial equivalent of tubulin.

The poliovirus replication complex involves the viral RNA-dependent RNA polymerase in oligomeric complexes on the surface of membranes. The building blocks of these oligomers are polymerase-polymerase interactions. One polymerase-polymerase interaction site, Interface I, has been identified and validated by previous work (Structure 5:1109; EMBO 20:1153, JBC 277:31551). Interface I is an asymmetric interaction which forms extendable head to tail fibers of polymerase. Interestingly, purified polymerase forms large planar lattices, suggesting that a second oligomeric interface exists on the polymerase which stacks up interface I fibers to form the observed two-dimensional planar arrays. Identification of this second interaction site remains an open question. To generate plausible hypotheses about this second oligomeric interface, computational modelling was employed.

The polymerase undergoes an allosteric change upon forming interface I (EMBO 23:3462). To model the alternative polymerase conformations sampled, low frequency harmonic oscillations were calculated using normal mode analysis. These conformations were modelled into polymerase-polymerase fibers, giving rise to ten different conformations for a dimer along interface I. The surface convolution was calculated for each pair of fibers to generate all possible ways of combining the subunits, creating thousands of complexes.

The complexes were then clustered into groups according to the use of particular residues. Of the resulting classes of tetramer, only the parallel and anti-parallel sheets show potential for readily forming the observed two dimensional lattices. Selection of the symmetric and parsimonious interfaces within the family of parallel and anti-parallel sheets resulted in four candidate interfaces involving distinct patches of residues on the polymerase. Multiple sequence alignment of every known picornavirus polymerase exhibits high conservation and co-variation within the regions that make up the postulated second interface. Mutant polymerase designed to disrupt the positied interactions will be purified and turbidity assays will be performed for analysis of polymerization kinetics.