International Journal of Biological Macromolecules 46 (2010) 317–323 Contents lists available at ScienceDirect International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac Recognition of active and inactive catalytic triads: A template based approach Vikas Gupta a,1, N.A. Udaya Prakash a,1, V. Lakshmi b, R. Boopathy b, J. Jeyakanthan c, D. Velmurugan d, K. Sekar a,∗ a Bioinformatics Centre, (Centre of Excellence in Structural Biology and Bio-computing), Indian Institute of Science, Bangalore 560 012, India b School of Biotechnology and Genetic Engineering, Bharathiar University, Coimbatore 641 046, Tamil Nadu, India c National Synchrotron Radiation Research Center, 101 Hsin-Ann Road, Hsinchu Science Park, Hsinchu 30076, Taiwan d Department of Crystallography and Biophysics, University of Madras, Guindy Campus, Chennai 600 025, Tamil Nadu, India article info abstract Article history: It is well established that a sequence template along with the database is a powerful tool for identifying the Received 30 November 2009 biological function of proteins. Here, we describe a method for predicting the catalytic nature of certain Received in revised form 13 January 2010 proteins among the several protein structures deposited in the Protein Data Bank (PDB). For the present Accepted 14 January 2010 study, we considered a catalytic triad template (Ser-His-Asp) found in serine proteases. We found that a Available online 25 January 2010 geometrically optimized active site template can be used as a highly selective tool for differentiating an active protein among several inactive proteins, based on their Ser-His-Asp interactions. For any protein Keywords: to be proteolytic in nature, the bond angle between Ser O␥–Ser H␥···His N␧2 in the catalytic triad needs Catalytic triad ◦ ◦ ␥··· ␧2 Serine protease to be between 115 and 140 . The hydrogen bond distance between Ser H His N is more flexible in ␦1··· ␦1 Hydrogen bond network nature and it varies from 2.0 Å to 2.7 Å while in the case of His H Asp O , it is from 1.6 Å to 2.0 Å. 2 Three-dimensional structure In terms of solvent accessibility, most of the active proteins lie in the range of 10–16 Å , which enables Solvent accessibility easy accessibility to the substrate. These observations hold good for most catalytic triads and they can Intramolecular distances be employed to predict proteolytic nature of these catalytic triads. © 2010 Elsevier B.V. All rights reserved. 1. Introduction form catalytic cleavage of an appropriate bond of the substrate. Such a triad involvement was first identified in chymotrypsin and A protein’s sequence motifs and templates are important tools subtilisin [5,6], results in cleaving the peptide bond. Proteolytic for the identification or prediction of its biological function and enzymes are widely distributed in nature and mediate a wide range tertiary structure [1–3]. Normally, the templates are derived from of physiological responses from the onset of blood clotting [7] to the one-dimensional protein sequence signature by analyzing and the digestion [8] of proteins in the alimentary system. They also comparing the information from the known protein structures, play a major role in the tissue destruction associated with arthri- especially the data generated from sequence alignments and pat- tis, pancreatitis, and pulmonary emphysema. Such enzymes are tern matching techniques (for example [4]). Thus, the derived highly specific for its own substrates and the specificity is in terms three-dimensional template will provide a quantitative descrip- of identifying the residue of the substrate that fits into the bind- tion about the relative dispositions of the key residues in the active ing pocket of the enzyme, present immediately adjacent to scissile site of the enzyme based on their three-dimensional atomic coor- amide bond. A detailed explanation on the role of catalytic triad dinates. Such a template, when generated can be used to scan the has been published elsewhere, which is a comprehensive review databases of known protein structures to identify the catalytic cen- of the structural basis of substrate specificity in serine proteases ters. [9]. Another group has performed an extensive steric comparison The three residues of a catalytic triad (Ser-His-Asp of serine pro- of the catalytic site residues in serine proteases [10]. They analyzed tease), occur far apart in the primary structure of the enzyme, come the differences in the relative conformations of the Ser-His-Asp together in a specific conformation called the active site and per- residues by performing root mean square (rms) fits for all occur- rences. On the basis of the differences and similarities, they were able to classify the serine proteases into chymotrypsin and sub- stilisin families. They also found several examples of Ser-His-Asp ∗ Corresponding author at: Bioinformatics Centre, (Centre of Excellence in Struc- triads in non-proteolytic proteins in similar conformations to that tural Biology and Bio-computing), 101, Raman Building, Indian Institute of Science, of serine proteases [11]. Bangalore 560 012, Karnataka, India. Tel.: +91 80 22933059/22932469/23601409; Artymiuk et al. [12] have used graph-theory approach for fax: +91 80 23600683/23600551. E-mail address: [email protected] (K. Sekar). the identification of catalytic triad residues based on the three- 1 These authors equally contributed to this work. dimensional patterns of amino acid side chains in protein 0141-8130/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2010.01.011 318 V. Gupta et al. / International Journal of Biological Macromolecules 46 (2010) 317–323 structures. A different structural comparison of the serine proteases using a less specific technique has been performed by Fischer et al. [13]. Their method derived from geometric hashing is widely used in the field of computer vision research. In this method, all C␣ atoms in the backbone are considered as points in space and protein struc- tures are compared purely on the geometric relationships of these points. They were able to identify structural similarities between the active site of the trypsin-like and the subtilisin-like serine pro- teases based solely on the similarities of C␣ geometries of their constituent catalytic residues. Further, Cai et al. have employed support vector machines to identify catalytic triads present in ser- ine hydrolase family [14]. A method called Comparative Molecular Field Analysis (CoMFA) which provides statistical and graphical models that relate the properties of molecules (including biological activity) to their structures. These models are then used to predict the properties or activity of the novel compounds [15]. Apart from the serine proteases, the Ser-His-Asp catalytic triad also occurs in several lipases, which are responsible for hydrolyzing triglycerides into diglycerides and subsequently monoglycerides and free fatty acids. The catalytic mechanism in such lipases is also mediated by the catalytic serine of the triad [8,16]. The catalytic triad is buried beneath a short stretch of helix, known as the “lid”. A number of crystallographic studies have confirmed the hypothe- sis that the lid is displaced during activation of lipases [16,17], being rolled back as a rigid body into the hydrophilic trench filled previ- ously by water molecules, thereby exposing the active site. Thus, the present study is carried out to better understand the relative conformational orientation of the catalytic triad residues. Our anal- ysis differs substantially from the earlier studies [10,12–15].We aim to throw light on the geometric and physiochemical features of the catalytic triad of serine proteases and employ the results to differentiate between active and inactive catalytic triads. Thus, we derived templates of catalytic triads that are present in proteases. Further, we analyzed all protein structures in which the Ser O␥ atom and the Asp O␦1 and O␦2 atoms are present at a distance of 3.6 Å or less with the N␦1 and N␧2 atoms of the histidine residue. The hydro- gen bond networks and the orientation of serine, aspartate and histidine residues of the catalytic triad were also analyzed. Further, we have demonstrated that once a three-dimensional template is derived from the PDB file, it can be employed to provide valuable information for predicting the catalytic nature of a protein. 2. Materials and methods A total of 58,083 three-dimensional structures (as of June, 2009) were deposited in the PDB [18]. About 4655 structures in the PDB contain interactions between the three (Ser-His-Asp) amino acid residues. Among the residues present in the catalytic triad, a cut-off distance of 3.6 Å was considered between the Ser O␥ atom and Asp Fig. 1. (a) The ball and stick representation of the active catalytic triad identified O␦1 or O␦2 atom with His N␦1 or N␧2 atom. In about 2878 structures, in plasminogen activator protein (PDB-id: 1C5S). Interactions between Ser O –Ser H ···His Nε2 and His Nı1–His Hı1Asp···Oı2 atoms are shown. (b) The ball and stick the Ser O␥ atom was found to be hydrogen bonded with N␧2 of the representation of the active catalytic triad identified in Phospholipase A2 (PDB-id: histidine residue (Fig. 1a) and 1777 structures were found to have 1SQZ). The interactions between Ser O␥–Ser H␥–His N␦1 and HisN␧2–His H␧2···Asp ␥ ␦ interaction between Ser O and N 1 of the histidine residue (Fig. 1b). O␦2 atoms are shown. The histidine residue has an imidazole ring in which the hydrogen atom can shuttle from N␦1 to N␧2 and can be present in any of the two nitrogen atoms. Out of the total number of structures (4655) in further divided into two groups (group 1 proteins contain a cat- the dataset, 1131 were considered as false positives since they con- alytic triad and participate in proteolysis and group 2 proteins tain the Ser-His-Asp motif and have no associated catalytic activity.