David Barford

The structure of the tetratricopeptide repeats of protein phosphatase 5: implications for TPR-mediated protein-protein interactions.

The tetratricopeptide repeat (TPR) is a degenerate 34 amino acid sequence identified in a wide variety of proteins, present in tandem arrays of 3–16 motifs, which form scaffolds to mediate protein–protein interactions and often the assembly of multiprotein complexes. TPR‐containing proteins include the anaphase promoting complex (APC) subunits cdc16, cdc23 and cdc27, the NADPH oxidase subunit p67 phox, hsp90‐binding immunophilins, transcription factors, the PKR protein kinase inhibitor, and peroxisomal and mitochondrial import proteins. Here, we report the crystal structure of the TPR domain of a protein phosphatase, PP5. Each of the three TPR motifs of this domain consist of a pair of antiparallel α‐helices of equivalent length. Adjacent TPR motifs are packed together in a parallel arrangement such that a tandem TPR motif structure is composed of a regular series of antiparallel α‐helices. The uniform angular and spatial arrangement of neighbouring α‐helices defines a helical structure and creates an amphipathic groove. Multiple‐TPR motif proteins would fold into a right‐handed super‐helical structure with a continuous helical groove suitable for the recognition of target proteins, hence defining a novel mechanism for protein recognition. The spatial arrangement of α‐helices in the PP5–TPR domain is similar to those within 14‐3‐3 proteins.

Structural basis for the recognition of regulatory subunits by the catalytic subunit of protein phosphatase 1

The diverse forms of protein phosphatase 1 in vivo result from the association of its catalytic subunit (PP1c) with different regulatory subunits, one of which is the G‐subunit (GM) that targets PP1c to glycogen particles in muscle. Here we report the structure, at 3.0 Å resolution, of PP1c in complex with a 13 residue peptide (GM[63–75]) of GM. The residues in GM[63–75] that interact with PP1c are those in the Arg/Lys–Val/Ile–Xaa–Phe motif that is present in almost every other identified mammalian PP1‐binding subunit. Disrupting this motif in the GM[63–75] peptide and the M110[1–38] peptide (which mimics the myofibrillar targeting M110 subunit in stimulating the dephosphorylation of myosin) prevents these peptides from interacting with PP1. A short peptide from the PP1‐binding protein p53BP2 that contains the RVXF motif also interacts with PP1c. These findings identify a recognition site on PP1c, invariant from yeast to humans, for a critical structural motif on regulatory subunits. This explains why the binding of PP1 to its regulatory subunits is mutually exclusive, and suggests a novel approach for identifying the functions of PP1‐binding proteins whose roles are unknown.

The Structure of the Protein Phosphatase 2A PR65/A Subunit Reveals the Conformation of Its 15 Tandemly Repeated HEAT Motifs

The PR65/A subunit of protein phosphatase 2A serves as a scaffolding molecule to coordinate the assembly of the catalytic subunit and a variable regulatory B subunit, generating functionally diverse heterotrimers. Mutations of the beta isoform of PR65 are associated with lung and colon tumors. The crystal structure of the PR65/Aalpha subunit, at 2.3 A resolution, reveals the conformation of its 15 tandemly repeated HEAT sequences, degenerate motifs of approximately 39 amino acids present in a variety of proteins, including huntingtin and importin beta. Individual motifs are composed of a pair of antiparallel alpha helices that assemble in a mainly linear, repetitive fashion to form an elongated molecule characterized by a double layer of alpha helices. Left-handed rotations at three interrepeat interfaces generate a novel left-hand superhelical conformation. The protein interaction interface is formed from the intrarepeat turns that are aligned to form a continuous ridge.

The crystal structure of human eukaryotic release factor eRF1--mechanism of stop codon recognition and peptidyl-tRNA hydrolysis.

The release factor eRF1 terminates protein biosynthesis by recognizing stop codons at the A site of the ribosome and stimulating peptidyl-tRNA bond hydrolysis at the peptidyl transferase center. The crystal structure of human eRF1 to 2.8 A resolution, combined with mutagenesis analyses of the universal GGQ motif, reveals the molecular mechanism of release factor activity. The overall shape and dimensions of eRF1 resemble a tRNA molecule with domains 1, 2, and 3 of eRF1 corresponding to the anticodon loop, aminoacyl acceptor stem, and T stem of a tRNA molecule, respectively. The position of the essential GGQ motif at an exposed tip of domain 2 suggests that the Gln residue coordinates a water molecule to mediate the hydrolytic activity at the peptidyl transferase center. A conserved groove on domain 1, 80 A from the GGQ motif, is proposed to form the codon recognition site.

Crystal structure of the protein serine/threonine phosphatase 2C at 2.0 A resolution.

Protein phosphatase 2C (PP2C) is a Mn2+‐ or Mg2+‐dependent protein Ser/Thr phosphatase that is essential for regulating cellular stress responses in eukaryotes. The crystal structure of human PP2C reveals a novel protein fold with a catalytic domain composed of a central beta‐sandwich that binds two manganese ions, which is surrounded by alpha‐helices. Mn2+‐bound water molecules at the binuclear metal centre coordinate the phosphate group of the substrate and provide a nucleophile and general acid in the dephosphorylation reaction. Our model presents a framework for understanding not only the classical Mn2+/Mg2+‐dependent protein phosphatases but also the sequence‐related domains of mitochondrial pyruvate dehydrogenase phosphatase, the Bacillus subtilus phosphatase SpoIIE and a 300‐residue domain within yeast adenyl cyclase. The protein architecture and deduced catalytic mechanism are strikingly similar to the PP1, PP2A, PP2B family of protein Ser/Thr phosphatases, with which PP2C shares no sequence similarity, suggestive of convergent evolution of protein Ser/Thr phosphatases.

TOPOLOGICAL CHARACTERISTICS OF HELICAL REPEAT PROTEINS

The recent elucidation of protein structures based upon repeating amino acid motifs, including the armadillo motif, the HEAT motif and tetratricopeptide repeats, reveals that they belong to the class of helical repeat proteins. These proteins share the common property of being assembled from tandem repeats of an alpha-helical structural unit, creating extended superhelical structures that are ideally suited to create a protein recognition interface.

PTPN11 (Shp2) mutations in LEOPARD syndrome have dominant negative, not activating, effects

Multiple lentigines/LEOPARD syndrome (LS) is a rare, autosomal dominant disorder characterized by Lentigines, Electrocardiogram abnormalities, Ocular hypertelorism, Pulmonic valvular stenosis, Abnormalities of genitalia, Retardation of growth, and Deafness. Like the more common Noonan syndrome (NS), LS is caused by germ line missense mutations in PTPN11, encoding the protein-tyrosine phosphatase Shp2. Enzymologic, structural, cell biological, and mouse genetic studies indicate that NS is caused by gain-of-function PTPN11 mutations. Because NS and LS share several features, LS has been viewed as an NS variant. We examined a panel of LS mutants, including the two most common alleles. Surprisingly, we found that in marked contrast to NS, LS mutants are catalytically defective and act as dominant negative mutations that interfere with growth factor/Erk-mitogen-activated protein kinasemediated signaling. Molecular modeling and biochemical studies suggest that LS mutations contort the Shp2 catalytic domain and result in open, inactive forms of Shp2. Our results establish that the pathogenesis of LS and NS is distinct and suggest that these disorders should be distinguished by mutational analysis rather than clinical presentation.

Revealing mechanisms for SH2 domain mediated regulation of the protein tyrosine phosphatase SHP-2

The crystal structure of the protein tyrosine phosphatase SHP-2 reveals the mechanism of auto-inhibition of phosphatase activity by its SH2 domains. Phosphotyrosine peptide stimulation of the phosphatase activity, resulting from peptide binding to the N-terminal SH2 domain, is linked to conformational changes within the protein, including an unprecedented allosteric transition of the N-terminal SH2 domain.

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

Protein-tyrosine phosphatases (PTPs) are signal transduction enzymes that catalyze the dephosphorylation of phosphotyrosine residues via the formation of a transient cysteinyl-phosphate intermediate. The mechanism of hydrolysis of this intermediate has been examined by generating a Gln-262 → Ala mutant of PTP1B, which allows the accumulation and trapping of the intermediate within a PTP1B crystal. The structure of the intermediate at 2.5-Å resolution reveals that a conformationally flexible loop (the WPD loop) is closed over the entrance to the catalytic site, sequestering the phosphocysteine intermediate and catalytic site water molecules and preventing nonspecific phosphoryltransfer reactions to extraneous phosphoryl acceptors. One of the catalytic site water molecules, the likely nucleophile, forms a hydrogen bond to the putative catalytic base, Asp-181. In the wild-type enzyme, the nucleophilic water molecule would be coordinated by the side chain of Gln-262. In combination with our previous structural data, we can now visualize each of the reaction steps of the PTP catalytic pathway. The hydrolysis of the cysteinyl-phosphate intermediate of PTPs is reminiscent of GTP hydrolysis by the GTPases, in that both families of enzymes utilize an invariant Gln residue to coordinate the attacking nucleophilic water molecule.

Protein tyrosine phosphatases take off.

Protein tyrosine phosphatases (PTPs) are a family of signal transduction enzymes that dephosphorylate phosphotyrosine containing proteins. Structural and kinetic studies provide a molecular understanding of how these enzymes regulate a wide range of intracellular processes.