Predicting the Structure of the Flavodoxin fromEschericia coliby Homology Modeling, Distance Geometry and Molecular Dynamics

Abstract

Abstract As part of our on-going development of a method, based upon distance geometry calculations, for predicting the structures of proteins from the known structures of their homologues, we have predicted the structure of the 176 residue Flavodoxin from Escherichia coli. This prediction was based upon the crystal structures of the homologous Flavodoxins from Anacystis nidulans, Chondrus crispus, Desulfovibrio vulgaris and Clostridium beijerinckii, whose sequence identities with Escherichia coli were 44%, 33%, 23% and 16%, respectively. A total of 13,043 distance constraints among the alpha-carbons of the Escherichia coli structure were derived from the sequence alignments with the known structures, together with 8,893 distance constraints among backbone and sidechain atoms of adjacent residues, 978 between the alpha-carbons and selected atoms of the flavin mononucleotide cofactor, 116 constraints to enforce conserved hydrogen bonds, and 452 constraints on the torsion angles in conserved residues. An ensemble of ten random Escherichia coli structures was computed from these constraints, with an average root mean square coordinate deviation (RMSD) among the alpha carbons of 0.85 Ångstroms (excluding the first 1 and last 6 residues, which have no corresponding residues in any of the homologues and hence were unconstrained); the corresponding average heavy-atom RMSD was 1.60 Å. Since the distance geometry calculations were performed without hydrogen atoms, protons were added to the resulting structures and these structures embedded in a 50 × 50 × 40 Å solvent box with periodic boundary conditions. They were then subjected to a 20 picosecond dynamical simulated annealing procedure, starting at 300 K and gradually reduced to 10K, in which all the distance and torsion angle constraints were maintained by means of harmonic restraint functions. This was followed up by 1000 iterations of unrestrained conjugate gradients minimization. The goal of this energy refinement procedure was not to drastically modify the structures in an attempt at a priori prediction, but merely to improve upon the predictions obtained from the geometric constraints, particularly with regard to their local backbone and sidechain conformations and their hydrogen bonds. The resulting structures differed from the respective starting structures by an average of 1.52 Å in their heavy atom RMSD's, while the average RMSD among the heavy atoms of residues 2-170 increased slightly to 1.66 Å. We hope these structures will be good enough to enable the phase problem to be solved for the crystallographic data that is now being collected on this protein.