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Theoretical model of restriction endonuclease HpaI in complex with DNA, predicted by fold recognition and validated by site‐directed mutagenesis
Author(s) -
Skowronek Krzysztof J.,
Kosinski Jan,
Bujnicki Janusz M.
Publication year - 2006
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.20920
Subject(s) - restriction enzyme , computational biology , mutagenesis , protein engineering , threading (protein sequence) , protein structure prediction , genetics , dna , biology , restriction site , mutant , protein structure , biochemistry , enzyme , gene
Type II restriction enzymes are commercially important deoxyribonucleases and very attractive targets for protein engineering of new specificities. At the same time they are a very challenging test bed for protein structure prediction methods. Typically, enzymes that recognize different sequences show little or no amino acid sequence similarity to each other and to other proteins. Based on crystallographic analyses that revealed the same PD‐(D/E)XK fold for more than a dozen case studies, they were nevertheless considered to be related until the combination of bioinformatics and mutational analyses has demonstrated that some of these proteins belong to other, unrelated folds PLD, HNH, and GIY‐YIG. As a part of a large‐scale project aiming at identification of a three‐dimensional fold for all type II REases with known sequences (currently ∼ 1000 proteins), we carried out preliminary structure prediction and selected candidates for experimental validation. Here, we present the analysis of HpaI REase, an ORFan with no detectable homologs, for which we detected a structural template by protein fold recognition, constructed a model using the FRankenstein monster approach and identified a number of residues important for the DNA binding and catalysis. These predictions were confirmed by site‐directed mutagenesis and in vitro analysis of the mutant proteins. The experimentally validated model of HpaI will serve as a low‐resolution structural platform for evolutionary considerations in the subgroup of blunt‐cutting REases with different specificities. The research protocol developed in the course of this work represents a streamlined version of the previously used techniques and can be used in a high‐throughput fashion to build and validate models for other enzymes, especially ORFans that exhibit no sequence similarity to any other protein in the database. Proteins 2006. © 2006 Wiley‐Liss, Inc.