Visualization of the Cysteinyl-phosphate Intermediate of a Protein-tyrosine Phosphatase by X-ray Crystallography

Abstract

The formation of phosphoryl-enzyme intermediates is an essential component of numerous enzymatic mechanisms that involve phosphoryl-transfer reactions, for example the dephosphorylation of Tyr(P) residues catalyzed by protein-tyrosine phosphatases (1). PTPs 1 together with the protein-tyrosine kinases, which catalyze the opposing tyrosine phosphorylation reaction, control the overall levels of cellular tyrosine phosphorylation, and the molecular basis of the regulation and substrate specificity of these enzymes is subject to much investigation (2, 3). Reversible tyrosine phosphorylation is essential to the signal transduction pathways triggered by hormones, mitogens, and oncogenes that regulate such processes as cell growth, differentiation, and proliferation. The PTPs are a diverse family of enzymes that comprise transmembrane receptor-like PTPs (RPTPs) and soluble cytosolic proteins. The catalytic domains of the PTPs are highly conserved, consisting of ϳ250 amino acids and are characterized by an 11-residue PTP signature motif, (I/V)HCXAGXXR(S/T)G, containing the Cys and Arg residues that are essential for catalysis (4, 5). Diversity within the family is generated by the nature of the noncatalytic segments attached to the N and C termini of the PTP domains, which provide regulatory and subcellular targeting functions. Additional diversity is generated within the RPTPs, which frequently possess tandem PTP domains, although for some RPTPs such as CD45, the C-terminal PTP domain lacks catalytic activity (3). The dual specificity phosphatases (DSPs) are related to the PTPs by their possession of the PTP signature motif and related tertiary structure and catalytic mechanism (1, 6).Insights into the catalytic mechanism of PTPs and DSPs have been obtained from structural and kinetic studies of these enzymes (1, 4, 5). A PTP1B-phosphotyrosine peptide complex revealed that the Tyr(P) residue of the peptide is buried within a deep catalytic site cleft present on the protein's molecular surface. The base of the catalytic site is formed by residues of the PTP signature motif with the phosphate group of Tyr(P) being coordinated by main-chain amide groups and the Arg side chain of this motif, such that the phosphorus atom is situated adjacent to the S␥ atom of the catalytic Cys residue (7). Four other loops bearing invariant residues form the sides of the catalytic cleft and contribute to catalysis and substrate recognition. Engagement of phosphopeptides by PTP1B promotes a major conformational change of one of these loops (the WPD loop) consisting of residues 179 -187 that shift by as much as 8 Å to close over the phenyl ring of Tyr(P) and allow the side chain of Asp-181 to act as a general acid in the catalytic reaction. The Arg-221 side chain reorients to optimize salt bridge interactions with the phosphate bound to the catalytic site. This shift is coupled to motion of the WPD loop via a hydrogen bond between NH 2 of Arg-221 and the carbonyl oxygen of Pro-180 and hydrophobic interactions between the aliphatic moiety of ...

The Peroxiredoxin Repair Proteins

“…This latter finding coupled with the structural similarity to protein tyrosine phosphatases (e.g. PTP1B) (Pannifer et al, 1998) suggests yet another putative intermediate, the phosphorylated form of Srx. In this comparison both Srx and PTP1B contain a conserved Arg residue within a phosphate-binding motif adjacent to an essential Cys residue.…”

Protein cysteine phosphorylation of SarA/MgrA family transcriptional regulators mediates bacterial virulence and antibiotic resistance

“…Despite the fact that cysteine is the most nucleophilic residue among natural amino acids and is known to be subject to multiple PTMs (56), Cys-phosphorylation was previously considered an unusual PTM with only a few known examples as catalytic intermediates in enzymatic reactions (24,57,58). We show here that the conserved Cys residue in the global transcriptional factors SarA, MgrA, and SarZ can be phosphorylated, which controls virulence and other properties of S. aureus.…”

Why nature really chose phosphate

“…These enzymes differ in sequence and topology, and also whether they are cytosolic or membrane bound, but, with the exception of the cdc25 phosphatases, which have one residue substitution, all contain the characteristic (H/V)CX 5 R(S/T) sequence (Kolmodin & Å qvist, 2001). These enzymes utilize a two-step ' ping-pong ' mechanism, in which a nucleophilic cysteine attacks the bound phosphate, allowing for leaving group departure in a concerted fashion, with the assistance of an active site aspartate that acts as a general acid, resulting in a covalently bound phosphoenzyme intermediate (Guan & Dixon, 1991 ;Pannifer et al 1998). In the second step, the same aspartate now changes role to a general base, activating a water molecule for nucleophilic attack on the bound phosphate, to give P i and the restored enzyme.…”