A Measure of the Promiscuity of Proteins and Characteristics of Residues in the Vicinity of the Catalytic Site That Regulate Promiscuity Sandeep Chakraborty, Basuthkar J. Rao* Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, India, Abstract Promiscuity, the basis for the evolution of new functions through ‘tinkering’ of residues in the vicinity of the catalytic site, is yet to be quantitatively defined. We present a computational method Promiscuity IndicesEstimator (PROMISE) - based on signatures derived from the spatial and electrostatic properties of the catalytic residues, to estimate the promiscuity (PromIndex) of proteins with known active site residues and 3D structure. PromIndex reflects the number of different active site signatures that have congruent matches in close proximity of its native catalytic site, the quality of the matches and difference in the enzymatic activity. Promiscuity in proteins is observed to follow a lognormal distribution (m = 0.28, s = 1.1 reduced chi-square = 3.0E-5). The PROMISE predicted promiscuous functions in any protein can serve as the starting point for directed evolution experiments. PROMISE ranks carboxypeptidase A and ribonuclease A amongst the more promiscuous proteins. We have also investigated the properties of the residues in the vicinity of the catalytic site that regulates its promiscuity. Linear regression establishes a weak correlation (R2,0.1) between certain properties of the residues (charge, polar, etc) in the neighborhood of the catalytic residues and PromIndex. A stronger relationship states that most proteins with high promiscuity have high percentages of charged and polar residues within a radius of 3 A˚ of the catalytic site, which is validated using one-tailed hypothesis tests (P-values,0.05). Since it is known that these characteristics are key factors in catalysis, their relationship with the promiscuity index cross validates the methodology of PROMISE. Citation: Chakraborty S, Rao BJ (2012) A Measure of the Promiscuity of Proteins and Characteristics of Residues in the Vicinity of the Catalytic Site That Regulate Promiscuity. PLoS ONE 7(2): e32011. doi:10.1371/journal.pone.0032011 Editor: Bin Xue, University of South Florida, United States of America Received October 19, 2011; Accepted January 19, 2012; Published February 16, 2012 Copyright: ß 2012 Chakraborty, Rao. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by the Tata Institute of Fundamental Research (Department of Atomic Energy). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] Introduction analysis of multispecific enzymes. ‘Rigorous and quantitative measures of promiscuity’ which will measure the ‘magnitude and Jensen first proposed that promiscuity shaped the evolution of degree of promiscuity in a wide range of proteins’ is thus required primitive cells which presumably had minimal gene content [1–3]. [8]. Biochemical ‘leakiness’/‘messiness’ supplemented by gene dupli- We present a computational method for assigning a relative cation ensured increased gene content and specialization [4,5]. promiscuous index (PromIndex) to proteins with known active site The catalysis of reactions distinct from the one the protein has and 3D structure - Promiscuity IndicesEstimator (PROMISE). evolved to perform, but using the same domain, is a definition of PROMISE is based on the previously described method for active promiscuity adopted by many researchers including the current site detection which relies on the spatial and electrostatic work [6–8]. Promiscuity is distinguished from moonlighting properties of the catalytic residues (CLASP) [19]. Modular functions which are typically catalyzed using a domain of the approaches similar to CLASP using only spatial congruence have protein different from the active site scaffold [9,10]. been previously adopted [20–24]. Pruning based on electrostatic Protein engineers tailor innovative proteins by honing an existing moonlighting or promiscuous activity [11–14], often properties done by CLASP reduces considerable false positives without compromising its native function [15]. Inspite of the compared to purely 3D matching method, as potential difference mushrooming number of promiscuous proteins being discovered, congruence implies an appropriate milieu in the catalytic site [19]. till date there is no quantitative measure of promiscuity of proteins. Another innovative method [25] tries to dock high-energy A recent study traces the evolution of domain promiscuity along intermediates of various metabolites listed in a database [26] various evolutionary pathways [16], while another method and successfully predicts the function of an unknown protein. proposed a numerical analysis of promiscuous domains from CLASP was applied on alkaline phosphatases (APs), one of the genomic sequences [17]. An attempt at quantifying promiscuity widely studied promiscuous enzymes [27], which led to the provides a measure of the catalytic efficiencies of an enzyme discovery of a promiscuous protease activity in shrimp AP [19], toward a pre-defined set of substrates, but is limited in its scope and a promiscuous metallo-beta-lactamase activity in E. Coli AP and scalability [18]. Since this method assumes a uniform (S. Chakraborty, R. Minda, L. Salaye, J.M. Frere, Basuthkar J. chemical transformation on all substrates, it is more apt for the Rao, Manuscript in preparation). PLoS ONE | www.plosone.org 1 February 2012 | Volume 7 | Issue 2 | e32011 Quantitative Measure of Promiscuity of Proteins A set of non-homologous proteins with known active sites and the PROMISE computed promiscuity. The PromIndex of each structures from the Catalytic Atlas Site (CSA) [28], ‘‘an excellent protein has been calculated for at least 30 values, i.e. each protein starting point for characterizing mechanistically diverse superfam- figures at least 30 times in the randomly selected sets used for ilies’’ [29], was supplemented to include other promiscuous calculating PromIndex. Figure 1B shows the PromIndex comput- proteins described in a recent review [8] (Sproteins: |Spro- ed on the full population (which is skewed with respect to the teins| = 305).. PromIndex is now computed for each protein in the representation of each EC number) as compared with the mean set reflecting the number of matching active site signatures from and median obtained from balanced sets used in Figure 1A. other proteins in the vicinity of the native catalytic site, the quality Figure 1A and Figure 1B have the same ordering of proteins - but of the matches and how much the EC numbers differ. Since the set Figure 1A is based on (many) balanced sets and Figure 1B is based of proteins is unbalanced with respect to the EC numbers, random on the full set. As can be seen, the indices follow the same trend - sample sets such that all Enzyme Commission (EC) numbers are and is not randomly scattered. At first glance it appears that this equally represented are created from Sproteins. Computation of imbalance biases the promiscuity - since there are more proteins PromIndex was repeated multiple times by such random sampling from EC 3 (hydrolases), we expect hydrolases to be more to obtain statistics like mean, median and standard deviation, and promiscuous in this computation. However, this bias can be PromIndex was fitted for a lognormal distribution. PROMISE partially negated by assigning more weightage to matches across ranks a carboxypeptidase A, a catechol 2,3-dioxygenase, a ECs. Thus when we consider a protein from EC 6, even a single phosphoenolpyruvate carboxykinase, a kynureninase and a extra match with a protein from EC 3 (which are more ribonuclease-A as the most promiscuous, and a ribonucleotide predominant in this set), will add more to the promiscuity index reductase, a lipase and a haloalkane dehalogenase as the least neutralizing the effect of the higher number of EC 3 proteins. It is promiscuous proteins in Sproteins. We also establish a weak linear seen that the value of the weights chosen has virtually no effect on relationship (R2,0.1) between PromIndex and certain character- PromIndex. This is expected since the PromIndex is normalized. istics (% of charged and polar residues) in the immediate Figure 1C shows the variation in PromIndex when the radius neighborhood of the catalytic residues. Since these properties of around the catalytic residues which is to be considered as part of residues are known to be intrinsic to catalysis, this relationship the active site is varied from 1–6 A˚ . Note that the proteins are cross validates the methodology to quantify promiscuity. While the sorted according to the PromIndex as computed for a radius of forward relationship is weak (i.e. high percentage of polar residues 5A˚ . Figure 1D shows the distribution of PromIndex for the whole does not necessarily imply high promiscuity), the reverse population. This was fitted for a lognormal distribution relationship is quite strong (i.e. if the percentage of polar residues . is low, we can say with high confidence that the protein is not very ÀÁ ~ -y ~ 2 2 promiscuous). We establish low P-values (,0.05) based on fxðÞx; m,s e xsHðÞ2p ,y lnðÞ x -mÞ 2s hypothesis testing for proportion to establish that proteins with high promiscuity mostly have high % of charged and polar and has m = 0.28, s = 1.1 and reduced chi-square of 3.0E-5. residues within a radius of 3 A˚ from the catalytic residues, and that Pearson’s chi-squared tests the goodness of fit of an observed proteins which have low % of charged and polar residues (again frequency distribution with a theoretical distribution, and a within a 3 A˚ radius) are rarely promiscuous. reduced chi-square statistic is the Pearson’s chi-squared divided PROMISE is an automated computational method for by the number of degrees of freedom.