BIOINFORMATICS ORIGINAL PAPER doi:10.1093/bioinformatics/btm481 Structural bioinformatics Four-Body Scoring Function for Mutagenesis

MOTIVATION There is a need for an efficient and accurate computational method to identify the effects of single- and multiple-residue mutations on the stability and reactivity of proteins. Such a method should ideally be consistent and yet applicable in a widespread manner, i.e. it should be applied to various proteins under the same parameter settings, and have good predictive power for all of them. RESULTS We develop a Delaunay tessellation-based four-body scoring function to predict the effects of single- and multiple-residue mutations on the stability and reactivity of proteins. We test our scoring function on sets of single-point mutations used by several previous studies. We also assemble a new, diverse set of 237 single- and multiple-residue mutations, from over 24 different publications. The four-body scoring function correctly predicted the changes to the stability of 169 out of 210 mutants (80.5%), and the changes to the reactivity of 17 out of 27 mutants (63%). For the mutants that had the changes in stability/reactivity quantified (using reaction rates, temperatures, etc.), an average Spearman rank correlation coefficient of 0.67 was achieved with the four-body scores. We also develop an efficient method for screening huge numbers of mutants of a protein, called combinatorial mutagenesis. In one study, 64 million mutants of a cold-shock nucleus binding domain protein 1CSQ, with six of its residues being changed to all possible (20) amino acids, were screened within a few hours on a PC, and all five stabilizing mutants reported were correctly identified as stabilizing by combinatorial mutagenesis.

[1]  T L Blundell,et al.  Prediction of the stability of protein mutants based on structural environment-dependent amino acid substitution and propensity tables. , 1997, Protein engineering.

[2]  M. Levitt,et al.  Energy functions that discriminate X-ray and near native folds from well-constructed decoys. , 1996, Journal of molecular biology.

[3]  Alexander Tropsha,et al.  Development of a four-body statistical pseudo-potential to discriminate native from non-native protein conformations , 2003, Bioinform..

[4]  T. Blundell,et al.  The three‐dimensional structures of mutants of porphobilinogen deaminase: Toward an understanding of the structural basis of acute intermittent porphyria , 1994, Protein science : a publication of the Protein Society.

[5]  Marianne Manchester,et al.  Complete mutagenesis of the HIV-1 protease , 1989, Nature.

[6]  Arlo Z. Randall,et al.  Prediction of protein stability changes for single‐site mutations using support vector machines , 2005, Proteins.

[7]  A. Agrawal,et al.  Role of the Property of C-Reactive Protein to Activate the Classical Pathway of Complement in Protecting Mice from Pneumococcal Infection1 , 2006, The Journal of Immunology.

[8]  F. S. Mathews,et al.  Active-site residues are critical for the folding and stability of methylamine dehydrogenase. , 2001, Protein engineering.

[9]  Xiaochun Ge,et al.  Preliminary study on the structural basis of the antifungal activity of a rice lipid transfer protein. , 2003, Protein engineering.

[10]  B. Stoddard,et al.  Computational Thermostabilization of an Enzyme , 2005, Science.

[11]  L. Serrano,et al.  Predicting changes in the stability of proteins and protein complexes: a study of more than 1000 mutations. , 2002, Journal of molecular biology.

[12]  C. Erwin,et al.  Effects of engineered salt bridges on the stability of subtilisin BPN'. , 1990, Protein engineering.

[13]  P. Munson,et al.  Statistical significance of hierarchical multi‐body potentials based on Delaunay tessellation and their application in sequence‐structure alignment , 1997, Protein science : a publication of the Protein Society.

[14]  Herbert Edelsbrunner,et al.  Geometry and Topology for Mesh Generation , 2001, Cambridge monographs on applied and computational mathematics.

[15]  G J Pielak,et al.  A genetic approach for identifying critical residues in the fingers and palm subdomains of HIV-1 reverse transcriptase. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[16]  R. Jernigan,et al.  Estimation of effective interresidue contact energies from protein crystal structures: quasi-chemical approximation , 1985 .

[17]  Iosif I. Vaisman,et al.  Delaunay Tessellation of Proteins: Four Body Nearest-Neighbor Propensities of Amino Acid Residues , 1996, J. Comput. Biol..

[18]  M. Sippl.  Calculation of conformational ensembles from potentials of mean force. An approach to the knowledge-based prediction of local structures in globular proteins. , 1990, Journal of molecular biology.

[19]  J. Sussman,et al.  X‐ray structure of human acid‐β‐glucosidase, the defective enzyme in Gaucher disease , 2003, EMBO reports.

[20]  Y. Yamagata,et al.  Positive Contribution of Hydration Structure on the Surface of Human Lysozyme to the Conformational Stability* , 2002, The Journal of Biological Chemistry.

[21]  D Gilis,et al.  Predicting protein stability changes upon mutation using database-derived potentials: solvent accessibility determines the importance of local versus non-local interactions along the sequence. , 1997, Journal of molecular biology.

[22]  A. Tropsha,et al.  Four-body potentials reveal protein-specific correlations to stability changes caused by hydrophobic core mutations. , 2001, Journal of molecular biology.

[23]  T Schlick,et al.  Lattice protein folding with two and four‐body statistical potentials , 2001, Proteins.

[24]  G. von Heijne,et al.  Alanine Insertion Scanning Mutagenesis of Lactose Permease Transmembrane Helices* , 1997, The Journal of Biological Chemistry.

[25]  K. Regnström,et al.  Residues Important for Radical Stability in Ribonucleotide Reductase from Escherichia coli(*) , 1995, The Journal of Biological Chemistry.

[26]  U. Heinemann,et al.  Crystal Structure and Site-directed Mutagenesis of Bacillus macerans Endo-1,31,4--glucanase (*) , 1995, The Journal of Biological Chemistry.

[27]  E. Gouaux,et al.  Reduction of membrane protein hydrophobicity by site-directed mutagenesis: introduction of multiple polar residues in helix D of bacteriorhodopsin. , 1997, Protein engineering.

[28]  K. Kong,et al.  Site-directed mutagenesis study on the roles of evolutionally conserved aspartic acid residues in human glutathione S-transferase P1-1. , 1993, Protein engineering.

[29]  K Schulten,et al.  VMD: visual molecular dynamics. , 1996, Journal of molecular graphics.

[30]  Majid Masso,et al.  Computational mutagenesis studies of protein structure‐function correlations , 2006, Proteins.

[31]  D. Bevan,et al.  Correlation between protein binding strength on immobilized metal affinity chromatography and the histidine-related protein surface structure. , 2006, Analytical chemistry.

[32]  U. Arnold,et al.  Probing the unfolding region of ribonuclease A by site-directed mutagenesis. , 2004, European journal of biochemistry.

[33]  N. Bonander,et al.  Crystal structure of the disulfide bond-deficient azurin mutant C3A/C26A: how important is the S-S bond for folding and stability? , 2000, European journal of biochemistry.

[34]  Akinori Sarai,et al.  ProTherm and ProNIT: thermodynamic databases for proteins and protein–nucleic acid interactions , 2005, Nucleic Acids Res..

[35]  J. Gavilanes,et al.  Assignment of the contribution of the tryptophan residues to the spectroscopic and functional properties of the ribotoxin α‐sarcin , 2000, Proteins.

[36]  B. Persson,et al.  Active site directed mutagenesis of 3 beta/17 beta-hydroxysteroid dehydrogenase establishes differential effects on short-chain dehydrogenase/reductase reactions. , 1997, Biochemistry.

[37]  Jack Snoeyink,et al.  Almost-Delaunay simplices: nearest neighbor relations for imprecise points , 2004, SODA '04.

[38]  M. Sippl.  Calculation of conformational ensembles from potentials of mena force , 1990 .

[39]  Y. Yamagata,et al.  Effect of foreign N-terminal residues on the conformational stability of human lysozyme. , 1999, European journal of biochemistry.

[40]  F. Schmid,et al.  In-vitro selection of highly stabilized protein variants with optimized surface. , 2001, Journal of molecular biology.

[41]  P. Greengard,et al.  Site-directed mutagenesis of amino acid residues of protein phosphatase 1 involved in catalysis and inhibitor binding. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[42]  P. Alexander,et al.  Structural Basis of Thermostability , 2002, The Journal of Biological Chemistry.

[43]  D. Fournier,et al.  The effect of engineered disulfide bonds on the stability of Drosophila melanogaster acetylcholinesterase , 2006, BMC Biochemistry.

[44]  Crystal structure of the disulfide bond-deficient azurin mutant C3A/C26A: how important is the S-S bond for folding and stability? , 2000 .

[45]  Iosif I. Vaisman,et al.  Compositional preferences in quadruplets of nearest neighbor residues in protein structures: statistical geometry analysis , 1998, Proceedings. IEEE International Joint Symposia on Intelligence and Systems (Cat. No.98EX174).