Protein surface analysis for function annotation in high-throughput structural genomics pipeline

Abstract

Structural genomics (SG) initiatives are expanding the universe of protein fold space by rapidly determining structures of proteins that were intentionally selected on the basis of low sequence similarity to proteins of known structure. Often these proteins have no associated biochemical or cellular functions. The SG success has resulted in an accelerated deposition of novel structures. In some cases the structural bioinformatics analysis applied to these novel structures has provided specific functional assignment. However, this approach has also uncovered limitations in the functional analysis of uncharacterized proteins using traditional sequence and backbone structure methodologies. A novel method, named pvSOAR (pocket and void Surface of Amino Acid Residues), of comparing the protein surfaces of geometrically defined pockets and voids was developed. pvSOAR was able to detect previously unrecognized and novel functional relationships between surface features of proteins. In this study, pvSOAR is applied to several structural genomics proteins. We examined the surfaces of YecM, BioH, and RpiB from Escherichia coli as well as the CBS domains from inosine-5'-monosphate dehydrogenase from Streptococcus pyogenes, conserved hypothetical protein Ta549 from Thermoplasm acidophilum, and CBS domain protein mt1622 from Methanobacterium thermoautotrophicum with the goal to infer information about their biochemical function.

Figure 1.

An overview of the pvSOAR search methodology using YecM from E. coli. The structure of YecM (A) is submitted to the CASTp Web server (B) for pocket and void identification. A surface is identified, in this case CASTp pocket 18, and selected as the query template (C, green). The query is submitted to the pvSOAR Web server (D) and statistically significant surfaces matches are returned: neurolysin (PDB ID 1i1i, CASTp ID 85) from R. norvegicus (E, red), thermolysin (PDB ID 1lnd, CASTp ID 47) from B. thermoproteolyticus (F, orange), and from lethal toxin factor (PDB ID 1j7n, CASTp ID 198) from B. anthracis (G, yellow). The conserved residues between the query and library surfaces are superimposed (H). Figures were generated using PyMol (DeLano 2002) and CASTpyMol plugin.

Figure 2.

The surface pocket (CASTp ID 20, orange) on BioH from E. coli (PDB ID 1m33) contains a grouping of basic residues (yellow) (A). The CoA binding from aminoglycoside 2′-N-acetyltransferase (AAC(2′)-lc) from M. tuberculosis (PDB ID 1m4g, E.C.2.3.1.-) (B). CoA ligand has been modeled into the surface of BioH (D), based on the superposition with AAC(2′)-lc (C). Using a smaller solvent probe radius (1.2 Å) to define the pocket, the surface reveals an additional channel protruding into the domain interface which contains the buried catalytic triad (yellow) and Phe¹⁴³ (magenta) (E).

Figure 3.

The surfaces containing the proposed catalytic residues of RpiB from E. coli (PDB ID 1nn4, CASTp ID 67) (A) and M. tuberculosis (PDB ID 1usl, CASTp ID 67) (B) and RpiA from E. coli (PDB ID 1o8b, CASTp ID 49) (C). The distribution of amino acid residues in each surface (D).

Figure 4.

Structures containing CBS domains from CBS domain protein mt1622 from M. thermoautotrophicum (PDB ID 1pbj) (A), inosine-5′-monosphate dehydrogenase (IMPDH) from S. pyogenes (PDB ID 1zfj, E.C.1.1.1.205) (Zhang et al. 1999) (B), and conserved hypothetical protein Ta549 from T. acidophilum (PDB ID 1pvm) (C). Surfaces are colored by element type. The proposed nucleotide bindings surface of mt1622 (CASTp ID 9) has AMP modeled into it based on the superposition to a flavoprotein (PDB ID 1efp) (A). The proposed nucleotide bindings surface of IMPDH (CASTp ID 31) has ATP modeled into it based on the superposition to cyclin-dependent kinase 2 (PDB ID 1b38) (B). Ta549 contains an additional C terminus CBS domain (C, orange) opposite the tandem domain interface surface (C, green). The domain insert creates a novel surface that shares similarity to an ATP binding surface from saicarsynthase (PDB ID 1obd). An ATP molecule has been modeled into the surface of Ta549 (orange) based on the superposition of the conserved residues (D). A stereo representation of the surfaces from Ta549 (orange) and saicarsynthase (white) with the modeled ATP (E).