Structural Studies of Transition-State Stabilization by a Small Catalytic RNA, Metabolite-Binding by a Regulatory RNA Sequence and the Mechanism of Action of Activation Induced Deaminase.

Abstract

Thesis (Ph.D.)--University of Rochester. School of Medicine and Dentistry. Dept. of Biochemistry and Biophysics, 2008.Structural knowledge of an enzyme’s architecture can help elucidate the details of how it performs its biological role at the molecular level. In a case study of the hairpin ribozyme, structures were solved to high resolution in complex with reaction-intermediate analogs revealing key chemical groups in catalytically-relevant configurations. Importantly, the non-bridging oxygens of the scissile phosphate were tightly restrained in a conformation apparently promoting an inductive effect to elevate the pKa of a crucial active-site nucleobase while also ameliorating the unfavorable buildup of negative charge at the leaving group during the phosphoryl-transfer reaction. Active-site water molecules were ascribed a new role in both geometric and electrostatic stabilization of the reaction intermediate. By comparison to proteinaceous ribonuclease structures harboring the same reaction-intermediate analog, the catalytic strategies employed by these two evolutionarily-disparate enzyme classes appear to be a case of convergent evolution. In a second investigation, the aptamer domain of a metabolite-sensing non-coding regulatory RNA sequence was analyzed. Construct design, initial crystallization and attempts to solve the structure are described followed by improved methods to prepare a homogeneous RNA-metabolite complex. The resulting complex was evaluated by dynamic light scattering as well as small angle X-ray scattering. Subsequent optimization of crystallization conditions and diffraction analyses are also reported. The final area of focus encompasses biochemical and computational efforts to probe the mechanism of action of activation induced deaminase (AID). AID is a member of the cytidine deaminase superfamily and functions in the immunoglobulin maturation processes of somatic hypermutation (SHM), gene conversion (GC) and class switch recombination (CSR). Despite considerable research, questions remain regarding how AID is targeted to its substrate(s), what other cellular cofactors are necessary for function and how its activity is regulated since dysfunction or misregulation of AID has the potential to cause cancer. Efforts to crystallize AID were complemented by computational modeling, which has provided the basis for targeted biochemical studies aimed at answering outstanding questions. Perspectives on the success and pitfalls of continued modeling are discussed in light of recent empirical structural information reported for related family members