Crystal structures of the apo form and a complex of human LMW-PTP with a phosphonic acid provide new evidence of a secondary site potentially related to the anchorage of natural substrates

Abstract

Low molecular weight protein tyrosine phosphatases (LMW-PTP, EC 3.1.3.48) are a family of single-domain enzymes with molecular weight up to 18 kDa, expressed in different tissues and considered attractive pharmacological targets for cancer chemotherapy. Despite this, few LMW-PTP inhibitors have been described to date, and the structural information on LMW-PTP druggable binding sites is scarce. In this study, a small series of phosphonic acids were designed based on a new crystallographic structure of LMW-PTP complexed with benzylsulfonic acid, determined at 2.1 Å. In silico docking was used as a tool to interpret the structural and enzyme kinetics data, as well as to design new analogs. From the synthesized series, two compounds were found to act as competitive inhibitors, with inhibition constants of 0.124 and 0.047 mM. We also report the 2.4 Å structure of another complex in which LMW-PTP is bound to benzylphosphonic acid, and a structure of apo LMW-PTP determined at 2.3 Å resolution. Although no appreciable conformation changes were observed, in the latter structures, amino acid residues from an expression tag were found bound to a hydrophobic region at the protein surface. This regions is neighbored by positively charged residues, adjacent to the active site pocket, suggesting that this region might be not a mere artefact of crystal contacts but an indication of a possible anchoring region for the natural substrate—which is a phosphorylated protein.

Introduction

Protein phosphorylation and dephosphorylation are the main post-translational modifications of proteins in eukaryotic cells.¹ These modifications are catalyzed by protein kinases and phosphatases that modify serine, threonine or tyrosine residues on different proteins, receptors, transcription factors and binding proteins, thus controlling their biological functions. Tyrosine phosphorylation occurs to a much smaller extent than threonine/serine phosphorylation, but it plays a pivotal role in cellular signaling processes.2, 3, 4, 5 The cellular level of the tyrosine phosphorylation is regulated by the opposing activity of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs).⁶ Therefore, these enzymes control fundamental physiological processes such as cell growth and differentiation, cell cycle, metabolism, cytoskeletal function, and immune response. Accordingly, deregulated activity of PTPs and PTKs is involved in the development of numerous inherited and acquired human diseases such as neurological and cardiovascular disorders, infections, diabetes, cancer, and autoimmunity.7, 8, 9

Alonso and co-workers¹⁰ demonstrated that human genome contains 107 genes encoding both experimentally verified PTPs and proteins with a domain homologous to the catalytic domain of these PTPs. Among these genes, 81 are predicted to be active protein phosphatases. Based on the primary structure of the catalytic domains and the amino acid used in the catalytic reaction, PTPs are subdivided into four evolutionarily distinct classes: I, II, III (cysteine-based PTPs) and IV (aspartate-based PTPs). Despite the three-dimensional structures of the catalytic domains of the cysteine-based PTPs are strikingly similar, they possess different topologies and their regulatory domains vary significantly.¹⁰

In spite of relatively limited sequence similarity, the most significant feature of the protein tyrosine phosphatase (PTP) superfamily is the conservation of the signature motif CX₅R, which forms the phosphate-binding loop in the active site (known as the P-loop). This structurally conserved arrangement ensures an identical catalytic mechanism where the cysteine and the conserved arginine residues at catalytic site remain in close proximity to hold the phosphate group of the substrate in a position for nucleophilic attack by the cysteine thiol nucleophile.¹¹

Low molecular weight protein tyrosine phosphatases (LMW-PTP, EC 3.1.3.48) are a family of enzymes expressed in different tissues with molecular weight up to 18 kDa. They are single-domain enzymes and have been identified in a wide variety of organisms including rat, human, bovine, bacteria, yeast and plants.12, 13, 14, 15, 16, 17, 18, 19 In humans, Class II cysteine-based PTPs are represented by the members of LMW-PTP family (also known as acid phosphatase locus 1, ACP1), which are widely expressed with no particular tissue-specific expression. Four different human LMW-PTP messenger RNA, derived by alternative splicing of a single transcript, have been characterized. Two of them correspond to the classical active isoforms 1 (IF1, PTPfast/isoform F or HCPTPA) and 2 (IF2, PTPslow/isoform S or HCPTPB).²⁰ Both isoforms are single polypeptide chains of equal length which display difference only in a short sequence segment that corresponds to amino acid residues 40–73 in the mature protein. However, these isoforms present divergence in their physical chemistry properties, especially with respect to kinetics and consequently physiological functions.20, 21, 22, 23

In recent years, PTPs have gained considerable attention as important drug targets.²⁴ However, despite potential inhibitors have been designed, there are challenges in developing successful LMW-PTP inhibitors. Firstly, the low bioavailability and it is very common the observation of reactive oxygen species (ROS) production by reported PTPase inhibitors, with a consequent PTP inhibition occurring by indirect and nonspecific ways.4, 6, 10, 12, 25, 26, 27 Since LMW-PTP is proposed as a pharmacological target for cancer chemotherapy,9, 28 it is important to better understand the mechanism and mode of binding of its inhibitors.

LMW-PTP (wild type or mutated) crystallographic structures of a wide range of organisms were reported, most of them presenting ions or other chemical substances contained in the sample buffer, or the synthetic substrate p-nitrophenyl phosphate (pNPP).3, 13, 29, 30, 31, 32, 33, 34 In the case of isoform A human enzyme, structural data are scarce, with two crystal structures of isoform A reported to date, one in which a molecule of 2-(N-morpholino)ethanesulfonic acid (MES buffering agent) was observed in the active site (PDB ID 5PNT³) and another deposited under the PDB ID 3N8I (unpublished Ref. 35), in which water molecules are observed in the active site pocket and a molecule of 1-naphtylacetic acid (NLA) is bound to a surface region of the protein.

In the present study, a series of compounds was characterized, and three new crystal structures of the isoform A (IF1) of LMW-PTP were determined. These structures comprise one apo LMW-PTP structure and two complexes with small molecule—one with the hydrolysis product of the protease inhibitor PMSF (phenylmethylsulfonyl fluoride) and other in complex with the PMSF analogous benzylphosphonic acid. In both cases, the structures revealed that these compounds bind non-covalently to the LMW-PTP catalytic site, in a very similar fashion as predicted for the natural pTyr substrate, therefore, acting as pTyr mimetic. Further, both in the apo structure and in the complex with benzylphosphonic acid, an unexpected crystallographic site diverse from the known active site is occupied by amino acid residues from the construct expression tag.