The crystal structure of the H‐ras oncogene protein p21 complexed to the slowly hydrolysing GTP analogue GppNp has been determined at 1.35 A resolution. 211 water molecules have been built into the electron density. The structure has been refined to a final R‐factor of 19.8% for all data between 6 A and 1.35 A. The binding sites of the nucleotide and the magnesium ion are revealed in high detail. For the stretch of amino acid residues 61‐65, the temperature factors of backbone atoms are four times the average value of 16.1 A2 due to the multiple conformations. In one of these conformations, the side chain of Gln61 makes contact with a water molecule, which is perfectly placed to be the nucleophile attacking the gamma‐phosphate of GTP. Based on this observation, we propose a mechanism for GTP hydrolysis involving mainly Gln61 and Glu63 as activating species for in‐line attack of water. Nucleophilic displacement is facilitated by hydrogen bonds from residues Thr35, Gly60 and Lys16. A mechanism for rate enhancement by GAP is also proposed.

The crystal structure of the guanine-nucleotide-binding domain of p21 (amino acids 1-166) complexed to the guanosine triphosphate analogue guanosine-5'-(beta, gamma-imido)triphosphate (GppNp) has been determined at a resolution of 2.6 A. The topological order of secondary structure elements is the same as that of the guanine-nucleotide-binding domain of bacterial elongation factor EF-Tu. Many interactions between nucleotide and protein have been identified. The effects of point mutations and the conservation of amino-acid sequence in the guanine-nucleotide-binding proteins are discussed.

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Chorismate mutase catalyzes a key step in the shikimate biosynthetic pathway towards phenylalanine and tyrosine. Curiously, the intracellular chorismate mutase of Mycobacterium tuberculosis (MtCM; Rv0948c) has poor activity and lacks prominent active-site residues. However, its catalytic efficiency increases 4100-fold on addition of DAHP synthase (MtDS; Rv2178c), another shikimate-pathway enzyme. The 2.35 Å crystal structure of the MtCM-MtDS complex bound to a transition-state analogue shows a central core formed by four MtDS subunits sandwiched between two MtCM dimers. Structural comparisons imply catalytic activation to be a consequence of the repositioning of MtCM active-site residues on binding to MtDS. The mutagenesis of the Cterminal extrusion of MtCM establishes conserved residues as part of the activation machinery. The chorismatemutase activity of the complex, but not of MtCM alone, is inhibited synergistically by phenylalanine and tyrosine. The complex formation thus endows the shikimate pathway of M. tuberculosis with an important regulatory feature. Experimental evidence suggests that such noncovalent enzyme complexes comprising an AroQ d subclass chorismate mutase like MtCM are abundant in the bacterial order Actinomycetales.

Overexpression of the epidermal growth factor (EGF) receptor (EGFR) in cancer cells correlates with tumor malignancy and poor prognosis for cancer patients. For this reason, the EGFR has become one of the main targets of anticancer therapies. Structural data obtained in the last few years have revealed the molecular mechanism for ligand-induced EGFR dimerization and subsequent signal transduction, and also how this signal is blocked by either monoclonal antibodies or small molecules. Nimotuzumab (also known as h-R3) is a humanized antibody that targets the EGFR and has been successful in the clinics. In this work, we report the crystal structure of the Fab fragment of Nimotuzumab, revealing some unique structural features in the heavy variable domain. Furthermore, competition assays show that Nimotuzumab binds to domain III of the extracellular region of the EGFR, within an area that overlaps with both the surface patch recognized by Cetuximab (another anti-EGFR antibody) and the binding site for EGF. A computer model of the Nimotuzumab-EGFR complex, constructed by docking and molecular dynamics simulations and supported by mutagenesis studies, unveils a novel mechanism of action, with Nimotuzumab blocking EGF binding while still allowing the receptor to adopt its active conformation, hence warranting a basal level of signaling. [Cancer Res 2009;69(14):5851-9]

Cholera is the prime example of blood-group-dependent diseases, with individuals of blood group O experiencing the most severe symptoms. The cholera toxin is the main suspect to cause this relationship. We report the high-resolution crystal structures (1.1–1.6 Å) of the native cholera toxin B-pentamer for both classical and El Tor biotypes, in complexes with relevant blood group determinants and a fragment of its primary receptor, the GM1 ganglioside. The blood group A determinant binds in the opposite orientation compared to previously published structures of the cholera toxin, whereas the blood group H determinant, characteristic of blood group O, binds in both orientations. H-determinants bind with higher affinity than A-determinants, as shown by surface plasmon resonance. Together, these findings suggest why blood group O is a risk factor for severe cholera.

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The three-dimensional structures and biochemical properties of two mutants of the G-domain (residues 1-166) of p21H-ras, p21 (G12D) and p21 (G12P), have been determined in the triphosphate-bound form using guanosine 5'-(beta,gamma-imido)triphosphate (GppNHp). They correspond to the most frequent oncogenic and the only nononcogenic mutation of Gly-12, respectively. The G12D mutation is the only mutant analyzed so far that crystallizes in a space group different from wild type, and the atomic model of the protein shows the most drastic changes of structure around the active site as compared to wild-type p21. This is due to the interactions of the aspartic acid side chain with Tyr-32, Gln-61, and the gamma-phosphate, which result in reduced mobility of these structural elements. The interaction between the carboxylate group of Asp-12 and the gamma-phosphate is mediated by a shared proton, which we show by 31P NMR measurements to exist in solution as well. The structure of p21 (G12P) is remarkably similar to that of wild-type p21 in the active site, including the position of the nucleophilic water. The pyrrolidine ring of Pro-12 points outward and seems to be responsible for the weaker affinity toward GAP (GTPase-activating protein) and the failure of GAP to stimulate GTP hydrolysis.