Structural basis for the broad substrate specificity of the human tyrosylprotein sulfotransferase-1

Abstract

Tyrosylprotein sulfotransferases (TPSTs) are enzymes that catalyze post-translational tyrosine sulfation of proteins. In humans, there are only two TPST isoforms, designated TPST1 and TPST2. In a previous study, we reported the crystal structure of TPST2, which revealed the catalytic mechanism of the tyrosine sulfation reaction. However, detailed molecular mechanisms underlying how TPSTs catalyse a variety of substrate proteins with different efficiencies and how TPSTs catalyze the sulfation of multiple tyrosine residues in a substrate protein remain unresolved. Here, we report two crystal structures of the human TPST1 complexed with two substrate peptides that are catalysed by human TPST1 with significantly different efficiencies. The distinct binding modes found in the two complexes provide insight into the sulfation mechanism for these substrates. The present study provides valuable information describing the molecular mechanism of post-translational protein modifications catalysed by TPSTs.

New tools for evaluating protein tyrosine sulphation: Tyrosyl Protein Sulphotransferases (TPSTs) are novel targets for RAF protein kinase inhibitors

“…Analysis of a variety of synthetic peptides and intact proteins confirms that TPST1 and TPST2 can also control site-specific sulphation on multiple tyrosine residues, which are often clustered, consistent with a processive mechanism of modification [7, 27], or directionally distributed towards the substrate N-terminus [20, 28]. Crystal structures of TPST1 complexed with substrate peptides that are sulphated with different efficiencies have also been reported, and comparative analysis suggests differential substrate preferences for acidic residues adjacent to the site of modification [24, 29]. Structural comparison suggests a shared catalytic mechanism and substrate-binding energetics, driven by charge-based dynamic interactions.…”

“…The chemical similarity between the phosphate donor ATP and PAPS, the universal sulphate donor, led us to investigate whether peptide tyrosyl sulphation could be detected using a high-throughput enzymatic procedure previously validated for phosphorylation catalysed by ATP-dependent kinases. To isolate pure, enzymatically active recombinant TPST1 and TPST2, both were expressed at high levels in bacteria, and refolded after purification from inclusion bodies using published ‘slow’ procedures suitable for structural and enzymatic analysis of TPST1 [24] and TPST2 [29]. The affinity of our TPST1 and TPST2 preparations for the PAPS co-factor was found to be almost identical to that previously reported, and we confirmed that TPST1 and TPST2 were folded and could bind to a variety of physiological and non-physiological ligands.…”

“…Rottlerin, GW305074X, suramin and RAF265 were built using Spartan16 (https://www.wavefun.com) and energy minimised using the Merck molecular forcefield. GOLD 5.2 (CCDC Software;) was used to dock molecules [51], with the binding site defined as 10 Å around the 5’ phosphorous atom of PAP, using coordinates from human TPST1 PDB ID: 5WRI [24]. A generic algorithm with ChemPLP as the fitness function [52] was used to generate 10 binding-modes per ligand in HS2ST.…”

“…TPSTs interact with the sulphate-donor cofactor PAPS and an appropriate (often acidic) tyrosine-containing protein substrate. Recent experiments suggest that TPST1 and TPST2 might function as homo or heterodimers [23, 24], providing regulatory opportunities for the control of site-specific sulphation amongst substrates. In general, tyrosine sulphation occurs in an acidic context in proteins and model substrates [2, 18, 24-26], although some, including the bioactive protein gastrin, lack acid residues adjacent to the site of sulphation [14].…”

“…Recent experiments suggest that TPST1 and TPST2 might function as homo or heterodimers [23, 24], providing regulatory opportunities for the control of site-specific sulphation amongst substrates. In general, tyrosine sulphation occurs in an acidic context in proteins and model substrates [2, 18, 24-26], although some, including the bioactive protein gastrin, lack acid residues adjacent to the site of sulphation [14]. Analysis of a variety of synthetic peptides and intact proteins confirms that TPST1 and TPST2 can also control site-specific sulphation on multiple tyrosine residues, which are often clustered, consistent with a processive mechanism of modification [7, 27], or directionally distributed towards the substrate N-terminus [20, 28].…”