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Abstract

Statistics based on the RCSB Protein Data Bank (PDB), show that the preferred methods for structure determination of proteins are X-Ray (~ 85%) and NMR (~14 %). However, for proteins showing solubility issues or producing crystal structures unsuitable for X-ray analysis, the Homology model has emerged as a successful alternative to the empirical methods.

Homology or comparative modeling is an efficient method for generating the tertiary structure of proteins when homologous proteins (similar sequence) are known. Starting from a template generated from the sequence of the target protein, queries (sequence template) to a protein database are made, and sequence alignments and coordinate assignments are used to identify the proteins similarity patterns. These patterns are then assembled to provide a initial structural model of the protein of interest. These models have been used   in several applications, for example, structural and dynamics studies of proteins using molecular dynamic simulation, study of the effect of sequence amino acid mutations on protein structures, and drug-discovery/drug-development studies using protein-ligand docking techniques.

Summary of CCB's Involvement

Analysis of PIP5K structure

In collaboration with Prof. Sergio Grinstein group (The Hospital for Sick Children), homology model techniques were applied in the structural analysis of phosphatidylinositol-4-phosphate 5-kinase (PIP5K). The study suggested that all PIP5K isoforms expose a sizable polycationic surface that is predicted to direct the kinases to negatively charged membranes

Structure of type II PIP4K and models of type I PIP5K ,  and  isoforms.
(a-d) The reported structure of type II PIP4K (a) and the predicted structures of the PIP5K,  and -90 isoforms produced by homology modeling are shown in an equivalent orientation with their putative membrane interacting face pointing towards the observer. The color surfaces are colored according to the range of electrostatic potential (red ( 10.0 kT/e)). The electrostatic surface potentials were computed using the continuum solvation model embodied in the Poisson-Boltzmann method and the adaptive Poisson–Boltzmann solver implemented in the APBS software. (e-h) The proteins are shown in an orientation corresponding to a 90º rotation from the orientations in (a-d), such that the presumed membrane-associated face of the proteins points downwards. The dipole moments (yellow arrows) were calculated directly from the atomic models and the atom partial charges using Protein Dipole Moments Server. The arrows were drawn at a magnification of 1.5 times.

Mutation analysis of chaperonin-like proteins BBS6, BBS10, and BBS12

In collaboration with Prof. Elise Heon group, homology model techniques were used to model proteins involved in Bardet Biedl syndrome 6 (BBS6), BBS10, and BBS12, which is a genetically heterogeneous pleiotropic disorder, contributed to comprehensive genotype-phenotype correlation analysis.

Homology modeling of CRYBA4

A novel cataract gene, CRYBA4, was identified by genetic analysis of a large Indian family with autosomal dominant cataract phenotype. Homology model was used to model the structure of the CRYBA4 protein and to study the role of the amino acid Phe94, highly conserved among species sequences ( see  picture) in the maintaining the native protein structure. This work was collaborated with Prof. Elise Héon group from the XXXXXXXXXX

Figure Homology modeling of CRYBA4.
(A) Depicted is the N-terminal domain of the predicted atomic structure of CRYBA4 built using the known structure. The protein backbone is drawn using a solid ribbon model. -helix, -strand and turn structures are colored in red, cyan and light green, respectively. Val36, Leu69 and Phe94 residues are shown in full atomic detail.  
(B) Environment of the Phe94 and Ser94 side chains in the modeled CRYBA4 3D structure. The side-chain of Phe94 and Ser94 are colored in blue and orange, respectively.  Residues whose atoms are within 6.0 Å from side-chain atoms of Phe94 are shown.  The ~100 Å3 cavity formed by replacing the bulky Phe 94 by Ser is outlined by the mesh colored in cyan.  This cavity is lined by the side chains of hydrophobic residues surrounding Ser94.  The cavity volume was computed using the Proshape software using a probe radius of 0.2Å, thereby approximating the cavity delimited by the molecular surface of the surrounding residues. 
(C) Portion of the CRYBA4 structure in the vicinity of residue 69, highlighting the differences in the -sheet backbone structures of the wt (Leu69) and mt (Pro69) proteins. The backbone nitrogen of Leu69 forms a hydrogen bond with the carbonyl group of Trp54, contributing to the stability the anti parallel -sheet structure.  This hydrogen bond cannot form in the Pro69 mutant. Its backbone nitrogen cannot act as a hydrogen bond donor, since it is covalently linked to the proline ring.