This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Ribonucleotide reductase (RNR) catalyzes the rate-limiting step of de novo DNA synthesis by reducing NDPs to dNDPs. Though much structural and biochemical data on non-catalytic subunits of eukaryotic RNR exist, specifically mouse Rnr2, and yeast Rnr2p?Rnr4p, until now there is no reported structure for any eukaryotic Rnr1. Using MAD data from BioCARS, we solved the yeast Rnr1 structure. During this proposal we will solve several cognate effector-substrate complex structures to address how substrates are selected by specific dNTPs binding at the effector site. The yeast RNR assembly involves association of Rnr2-Rnr4 with Rnr1. C-terminal peptides of Rnr2 and Rnr4 can disrupt this assembly, and a new class of anticancer and antiviral inhibitors is designed based on Rnr2 peptides. We will solve structures of Rnr1 complexed with peptidomimetic libraries and anticancer agents like clofarabine. In yeast RNR activity is controlled allosterically by ATP (upregulator) and dATP (downregulator). We will solve the structures of Rnr1 complexed with ATP and dATP to address this. Yeast RNR is also downregulated by Sml1, which binds Rnr1. The C-termini of Sml1 inhibits RNR activity with reduced potency. We will solve the Sml1 structure using MAD and Rnr1 complexed with Sml1-derived peptides and intact Sml1. We have constructed numerous mutants of Rnr1to study structure-function relationships. Several have been crystallized both in the native form and in complex with effector-substrate complexes. Though accepted that ABeta is involved in the pathogenesis of Alzheimer?s, there is no consensus on its atomic structure. We will obtain this using neutralizing antibodies (Abs). The structures of the Abs alone can be used with models of AB for docking studies, or better, AB?Ab complexes can provide a molecular basis for designing drugs against Alzheimer?s. We have crystallized an AB-recognizing Fab in apo form and complexed to a truncated A? peptide, and several other AB-recognizing Abs.
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