The MDM2 oncoprotein is a cellular inhibitor of the p53 tumor suppressor in that it can bind the transactivation domain of p53 and downregulate its ability to activate transcription. In certain cancers, MDM2 amplification is a common event and contributes to the inactivation of p53. The crystal structure of the 109-residue amino-terminal domain of MDM2 bound to a 15-residue transactivation domain peptide of p53 revealed that MDM2 has a deep hydrophobic cleft on which the p53 peptide binds as an amphipathic alpha helix. The interface relies on the steric complementarity between the MDM2 cleft and the hydrophobic face of the p53 alpha helix and, in particular, on a triad of p53 amino acids-Phe19, Trp23, and Leu26-which insert deep into the MDM2 cleft. These same p53 residues are also involved in transactivation, supporting the hypothesis that MDM2 inactivates p53 by concealing its transactivation domain. The structure also suggests that the amphipathic alpha helix may be a common structural motif in the binding of a diverse family of transactivation factors to the TATA-binding protein-associated factors.
Recent structural studies of epidermal growth factor receptor (EGFR) family extracellular regions have identified an unexpected mechanism for ligand-induced receptor dimerization that has important implications for activation and inhibition of these receptors. Here we describe the 2.8 angstroms resolution X-ray crystal structure of the antigen binding (Fab) fragment from cetuximab (Erbitux), an inhibitory anti-EGFR antibody, in complex with the soluble extracellular region of EGFR (sEGFR). The sEGFR is in the characteristic "autoinhibited" or "tethered" inactive configuration. Cetuximab interacts exclusively with domain III of sEGFR, partially occluding the ligand binding region on this domain and sterically preventing the receptor from adopting the extended conformation required for dimerization. We suggest that both these effects contribute to potent inhibition of EGFR activation.
Vaccinia DNA topoisomerase breaks and rejoins DNA strands through a DNA-(3'-phosphotyrosyl)-enzyme intermediate. A C-terminal catalytic domain, Topo(81-314), suffices for transesterification chemistry. The domain contains a constellation of five amino acids, conserved in all eukaryotic type IB topoisomerases, that catalyzes attack of the tyrosine nucleophile on the scissile phosphate. The structure of the catalytic domain, consisting of ten alpha helices and a three-strand beta sheet, resembles the catalytic domains of site-specific recombinases that act via a topoisomerase IB-like mechanism. The topoisomerase catalytic pentad is conserved in the tertiary structures of the recombinases despite scant sequence similarity overall. This implies that the catalytic domains of type IB topoisomerases and recombinases derive from a common ancestral strand transferase.
Streptokinase is a plasminogen activator widely used to treat patients with myocardial infarction. However, streptokinase is not a protease, and must first bind and interact with plasminogen to form an enzymatic complex. By measuring the binding of recombinant streptokinase fragments to plasminogen, we have sought, first, to identify a plasminogen binding region in streptokinase and, second, to explore the relation between binding (via this region) and the generation of a functional streptokinase--plasminogen activator complex. Recombinant streptokinase bound in a saturable and specific manner to human Glu-plasminogen with a dissociation constant of 4.2 x 10(-10) M. Recombinant streptokinase fragments spanning amino acids 1-127 and 1-253 could not be shown to bind to Glu-plasminogen, whereas fragments spanning amino acids 1-352, 120-352, and 244-414 bound tightly to plasminogen and each fragment completely inhibited the binding of full-length streptokinase to plasminogen. Although these latter streptokinase fragments formed a complex with plasminogen, enzymatic assays indicated that none of them was capable of generating an active site. When the streptokinase region shared by these three fragments, spanning residues 244-352, was expressed, it also bound plasminogen and competitively inhibited the formation of a functional plasminogen activator complex by full-length streptokinase. Taken together, these data indicate that streptokinase binds to plasminogen with high affinity, that a primary binding region for plasminogen is located within amino acids 244-352, and that binding via this region is necessary for the generation of a functional plasminogen activator complex.
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