Inhibition of the cell cycle is widely considered as a new approach toward treatment for diseases caused by unregulated cell proliferation, including cancer. Since cyclin-dependent kinases (CDKs) are key enzymes of cell cycle control, they are promissing targets for the design and discovery of drugs with antiproliferative activity. The detailed structural analysis of CDK2 can provide valuable information for the design of new ligands that can bind in the ATP binding pocket and inhibit CDK2 activity. For this objective, the crystal structures of human CDK2 apoenzyme and its ATP complex were refined to 1.8 and 1.9 A, respectively. The high-resolution refinement reveals 12 ordered water molecules in the ATP binding pocket of the apoenzyme and five ordered waters in that of the ATP complex. Despite a large number of hydrogen bonds between ATP-phosphates and CDK2, binding studies of cyclic AMP-dependent protein kinase with ATP analogues show that the triphosphate moiety contributes little and the adenine ring is most important for binding affinity. Our analysis of CDK2 structural data, hydration of residues in the binding pocket of the apoenzyme, flexibility of the ligand, and structural differences between the apoenzyme and CDK2-ATP complex provide an explanation for the results of earlier binding studies with ATP analogues and a basis for future inhibitor design.

Plasminogen activator inhibitor-1 (PAI-1) is unique among the serine proteinase inhibitors (serpins) in that it can adopt at least three different conformations (active, substrate and latent). We report the X-ray structure of a cleaved substrate variant of human PAI-1, which has a new beta-strand s4A formed by insertion of the amino-terminal portion of the reactive-site loop into beta-sheet A subsequent to cleavage. This is in contrast to the previous suggestion that the non-inhibitory function of substrate-type serpins is mainly due to an inability of the reactive-site loop to adopt this conformation. Comparison with the structure of latent PAI-1 provides insights into the molecular determinants responsible for the transition of the stressed active conformation to the thermostable latent conformation.

The crystal structure of the RNA dodecamer 5'-GGACUUUGGUCC-3' has been determined from X-ray diffraction data to 2.6 A resolution. This oligomer forms an asymmetric double helix in the crystal. Four consecutive non-Watson-Crick base-pairs are formed in the middle of the duplex including the first intrahelical U-U (or T-T) pairs observed in an oligonucleotide crystal structure. Two different conformations of U-U pairs are observed in the context of the surrounding sequence. One of these pairs is highly twisted, allowing a bound water to bridge across strands in the major groove. The crystal packing illustrates a new form of RNA helix-helix interaction.

Six-helix bundle (6HB) formation is an essential step for many viruses that rely on a class I fusion protein to enter a target cell and initiate replication. Because the binding modes of small molecule inhibitors of 6HB formation are largely unknown, precisely how they disrupt 6HB formation remains unclear, and structure-based design of improved inhibitors is thus seriously hampered. Here we present the high resolution crystal structure of TMC353121, a potent inhibitor of respiratory syncytial virus (RSV), bound at a hydrophobic pocket of the 6HB formed by amino acid residues from both HR1 and HR2 heptad-repeats. Binding of TMC353121 stabilizes the interaction of HR1 and HR2 in an alternate conformation of the 6HB, in which direct binding interactions are formed between TMC353121 and both HR1 and HR2. Rather than completely preventing 6HB formation, our data indicate that TMC353121 inhibits fusion by causing a local disturbance of the natural 6HB conformation.cocrystal structure | respiratory syncytial virus | TMC353121 | viral fusion T o allow the deposition of their nucleic acid genome into a host cell, and to initiate their replication cycle, enveloped viruses have evolved complex membrane fusion machinery that includes a fusion protein (1, 2). Based on structural similarity, the viral fusion proteins from different viruses have been grouped into three distinct classes: I, II, and III (3, 4). Prototypic trimeric class I fusion proteins include HIV-1 gp41, influenza hemagglutinin and the fusion proteins from paramyxoviruses. The fusion protein (F) of respiratory syncytial virus (RSV), a paramyxovirus belonging to the pneumovirinae subfamily, assembles into a homotrimer that is cleaved at two proximal furin cleavage sites during biosynthesis, priming the protein for membrane fusion. Proteolytic cleavage of the fusion protein precursor (F 0 ) yields two polypeptides, F 1 and F 2 , joined by a disulfide bridge (Fig. 1). F 1 consists of an N-terminal hydrophobic fusion peptide, followed by a first heptad-repeat (HR1), an intervening globular domain, and a second heptadrepeat (HR2), which itself is N-terminal to the viral transmembrane and cytoplasmic regions (3). Once fusion is triggered, dramatic refolding of the prefusion conformation of the viral fusion protein occurs. Functional and structural studies have provided evidence that a folding intermediate is formed that contains a coiled-coil structure of three HR1 heptad repeats (5-8). This intermediate allows the fusion peptide to be inserted into the plasma membrane of a target cell. In the final stage of membrane fusion, the HR1-CTC structure irreversibly refolds into a 6HB complex with three HR2 heptad-repeats, resulting in membrane merger and stable fusion pore formation (5-14). In many viruses that rely on class I fusion proteins, the central HR1 trimeric coiled-coil (HR1-CTC) contains a hydrophobic pocket in each of its three grooves that has been proposed as a potential drug binding site (9, 10).The therapeutic value of inhibiting 6HB formation was establ...

Trypsin mRNA from the grey fleshfly (Neobellieria bullata) was reverse transcribed and amplified by means of PCR. Two cDNA species of 600 bp and 800 bp were cloned and sequenced. The 3' end of the gene (300 bp) was amplified by means of the rapid-amplification-of-cDNA-ends method, cloned and sequenced. The deduced protein sequence of 254 amino acids exhibited 46 % identity to Drosophila trypsin and 32 % identity to Anophiline trypsin and Aedes trypsin. Three-dimensional models of Neobellieria trypsin and Drosophila trypsin were built and compared. Both models contain two domains of P-barrel sheets as was shown by means of X-ray crystallography of mammalian trypsin. The catalytic active site is composed of the canonical triad of His42, Asp87 and Ser182 whereas Asp176 sits at the bottom of the specificity pocket. Southern blot analysis suggested that Neobellieria trypsin is encoded by one gene. Northern blot analysis showed that an early trypsin transcript is found in the midgut of sugarfed females. This message disappeared after a liver meal, and was replaced by a late transcript. Injection of trypsin-modulating oostatic factor (TMOF) at 10-9M prevented the disappearance and the translation of the early transcript. TMOF did not prevent the appearance of the late transcript. However, in the presence of the hormone the late transcript was not translated. Thus, TMOF is the biological signal that terminates the translation of trypsin mRNA in the fleshfly gut and probably in the mosquito gut.

The crystal structure of the RNA dodecamer 5-GGCC(GAAA)GGCC-3 has been determined from x-ray diffraction data to 2.3-Å resolution. In the crystal, these oligomers form double helices around twofold symmetry axes. Four consecutive non-Watson-Crick base pairs make up an internal loop in the middle of the duplex, including sheared G⅐A pairs and novel asymmetric A⅐A pairs. This internal loop sequence produces a significant curvature and narrowing of the double helix. The helix is curved by 34؇ from end to end and the diameter is narrowed by 24% in the internal loop. A Mn 2؉ ion is bound directly to the N7 of the first guanine in the Watson-Crick region following the internal loop and the phosphate of the preceding residue. This Mn 2؉ location corresponds to a metal binding site observed in the hammerhead catalytic RNA.The study of RNA structure by NMR and x-ray crystallographic methods is currently flourishing due to both improvements in methods of synthesis and purification and the impetus provided by discoveries of new biological functions of RNA. A common element of RNA secondary structure is the internal loop, an interruption in double helical RNA by a series of bases that cannot form standard Watson-Crick pairs. Internal loops are found, for example, in ribosomal RNA, ribozymes, viroids, protein regulatory sites, and SELEX-evolved RNAs. Characterization of the three-dimensional structure of internal loops and their effect on the helices that bracket them is still in an early stage. The crystal structures of several RNA oligomers incorporating symmetric internal loops have been previously determined (1-4) and shown to have continuous base pairing with formation of U⅐G, U⅐C, and U⅐U non-Watson-Crick pairs. The helices containing these internal loops generally retain an A-form geometry; however, the presence of tandem U⅐C base pairs in one structure (1) induced a dramatic widening of the major groove from about 4 Å to about 8 Å. Perturbations in regular RNA helices by internal loops may be utilized by regulatory proteins to recognize specific RNA structures such as the rev-responsive element (RRE) (5) and the iron regulatory element (IRE) (6). Recently, it has been shown that the G⅐U mispair responsible for recognition and aminoacylation of tRNA Ala by its synthetase can be substituted by other non-Watson-Crick base pairs, implying that distortion in the helix induced by mispairing and not a particular sequence may be responsible for recognition (7).

The three-dimensional structure of staphylokinase has been determined at 1.8 A. The puntative site of interaction with plasminogen was identified and epitopes were mapped.

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