Active Site Detection by Spatial Conformity and Electrostatic Analysis—Unravelling a Proteolytic Function in Shrimp Alkaline Phosphatase

Abstract

Computational methods are increasingly gaining importance as an aid in identifying active sites. Mostly these methods tend to have structural information that supplement sequence conservation based analyses. Development of tools that compute electrostatic potentials has further improved our ability to better characterize the active site residues in proteins. We have described a computational methodology for detecting active sites based on structural and electrostatic conformity - C ata L ytic A ctive S ite P rediction (CLASP). In our pipelined model, physical 3D signature of any particular enzymatic function as defined by its active sites is used to obtain spatially congruent matches. While previous work has revealed that catalytic residues have large pKa deviations from standard values, we show that for a given enzymatic activity, electrostatic potential difference (PD) between analogous residue pairs in an active site taken from different proteins of the same family are similar. False positives in spatially congruent matches are further pruned by PD analysis where cognate pairs with large deviations are rejected. We first present the results of active site prediction by CLASP for two enzymatic activities - β-lactamases and serine proteases, two of the most extensively investigated enzymes. The results of CLASP analysis on motifs extracted from Catalytic Site Atlas (CSA) are also presented in order to demonstrate its ability to accurately classify any protein, putative or otherwise, with known structure. The source code and database is made available at www.sanchak.com/clasp/. Subsequently, we probed alkaline phosphatases (AP), one of the well known promiscuous enzymes, for additional activities. Such a search has led us to predict a hitherto unknown function of shrimp alkaline phosphatase (SAP), where the protein acts as a protease. Finally, we present experimental evidence of the prediction by CLASP by showing that SAP indeed has protease activity in vitro.

MEPPitope: spatial, electrostatic and secondary structure perturbations in the post-fusion Dengue virus envelope protein highlights known epitopes and conserved residues in the Zika virus

“…Further, electrostatic potential difference (EPD) 79, 80 was also shown to be conserved in cognate pairs of active site residues in these active sites 52, 81, 82 . Comparison of apo and holo structures quantifying the spatial and electrostatic perturbations after ligand binding was shown to identify critical catalytic residues in several enzymes 38 .…”

“…The APBS (version 1.4) parameters were set as described previously in 52. APBS writes out the electrostatic potential in dimensionless units of kT/e where k is Boltzmann’s constant, T is the temperature in K and e is the charge of an electron.…”

Dipeptidyl peptidase-IV inhibitors used in type-2 diabetes inhibit a phospholipase C: a case of promiscuous scaffolds in proteins

“…The CLASP package ( http://www.sanchak.com/clasp) used for querying these proteins using motifs from trypsin and DPP4 is written in Perl on Ubuntu 20 . Hardware requirements are modest - all results here are from a simple workstation (8GB ram), and runtimes for analyzing the ~5000 proteins was about 24 hours.…”

“…Adaptive Poisson-Boltzmann Solver (APBS) and PDB2PQR packages were used to calculate the potential difference between the reactive atoms of the corresponding proteins 57, 58 . The APBS parameters and electrostatic potential units were set as described previously in Chakraborty et al 20 . All protein structures were rendered by PyMol ( http://www.pymol.org/).…”

“…Previous work by our group has established the spatial and electrostatic congruence in cognate residue pairs of the active site in proteins with the same functionality (CLASP) 20, 21 . CLASP analysis indicated that the phosphoinositide-specific phospholipase C (PI-PLC) from Bacillus cereus has spatial and electrostatic congruence with a serine protease motif 22 .…”