Functional Regulation and Protein Structural Dynamics of Poliovirus 3c Protease

Open Access
Author:
Chan, Yan Mei
Graduate Program:
Chemistry
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
June 10, 2015
Committee Members:
  • David D Boehr, Dissertation Advisor
  • David D Boehr, Committee Chair
  • Squire J Booker, Committee Member
  • Emmanuel Hatzakis, Committee Member
  • Craig Eugene Cameron, Committee Member
Keywords:
  • Viruses
  • Poliovirus
  • Structural Dynamics
  • Functional Regulation
  • 3C Protease
  • Protein NMR
Abstract:
Viruses employ numerous strategies to maximize the use of their compact genomes. These strategies include the generation of multifunctional proteins, and post-translational processing to produce various protein products that are functionally distinct. Poliovirus (PV) 3C protease (3C) serves as an excellent example of how viruses utilize these strategies. 3C is a multifunctional protein with RNA binding and protease activities. Because of its essential role in viral replication, 3C has long been a pharmacological target for viral inhibition. The studies presented in this dissertation provide a novel understanding of the functional regulation and protein structural dynamics of PV 3C protease. We have performed a comprehensive study on the conformational dynamics of 3C across multiple timescales. Using numclear magnetic resonance (NMR) R2 relaxation dispersion studies of 3C reveal that the cleft around the protease active site is highly dynamic in the µs to ms timescale, possibly to accommodate for substrate binding and release. In the ps to ns timescale, NMR spin relaxation studies revealed that the majority of the backbone amide bond vectors are well-ordered. Binding of RNA did not alter the overall dynamics in either timescale, but there are structural differences induced by RNA near the protease active site. Analyses of dynamic chemical shifts from relaxation dispersion studies revealed that binding of RNA induces a different selection of conformations that may impact peptide binding. Interestingly, NMR chemical shift perturbation (CSP) analyses and fluorescent-based assays revealed that binding of RNA may have an effect on the peptide binding site and peptide binding may have an effect on RNA binding. These effects appear to be RNA and peptide dependent. There are also differences in the ternary complexes (protein: RNA: peptide) based on the order of ligand addition. The interplay between RNA and peptide binding may have radical implications in the regulation of protein functions essential for the different steps in viral replication. Viruses are intracellular parasites that utilize the subcellular machinery of their host cell for their own replication by remodeling the host’s intracellular membranes. Lipids with phosphatidylinositol-4-phosphate (PI4P) head groups have been found to be crucial for the formation of these replication organelles in numerous positive sense RNA viruses, such as PV. Lipid binding assays show that the PV protease 3C specifically binds to PI4P. Using NMR spectroscopy, we have identified a novel PI4P binding pocket on PV 3C using chemical shift perturbation analysis. Other phosphoinositides (PI) also appear to interact with this site or nearby sites on 3C. Protein variants with amino acid substitutions in the proposed binding site exhibit ten-fold higher dissociation constants compared to wild type protein as seen in fluorescent polarization studies. These data suggest that a new class of PIP binding domains has been identified. Furthermore, our NMR data suggest that the PIP binding site may be allosterically regulated with the RNA binding site. We have made the exciting discovery that the addition of just a few amino acid residues to the C-terminus of 3C dramatically alters PIP and RNA binding, and we propose that this finding has radical implications for the regulation of PIP binding, RNA binding, and viral polyprotein function. In PV, the 3C and 3D proteins can also exist as part of the 3CD polyprotein, which is functionally different from the mature 3C and 3D forms. Additional domain-domain contacts between 3C and 3D were not observed in existing crystal structures of 3CD. Efficient PIP and RNA binding to 3C only occurs when the C-terminal tail has been extended either through a hexahistidine tag (3C-CHis) or through the addition of the first few amino acids corresponding to the N-terminal residues of 3D (3C-D6 or 3C-D10). Proteolytic cleavage of 3C then serves as a switch for PIP and RNA binding; 3C in the context of 3CD can tightly bind PIP lipids and RNA, but mature 3C cannot. This C-terminal switch is both sequence and length dependent. NMR relaxation dispersion (µs to ms) and spin relaxation (ps to ns) studies revealed that the C-terminal residues do not lead to substantial changes in the overall structural dynamics of 3C. We proposed that there is an allosteric network connecting the C-terminus with the N-terminal helix, which is important for PIP and RNA binding. The studies presented in this dissertation are central for the understanding of the allosteric regulation and structural dynamics of the multifunctional 3C protease, and provide indispensable knowledge for understanding the 3C protease for drug and vaccine design purposes. This knowledge may have crucial implications for the understanding of both the regulation of post-translational processing pathways and the function of viral replication organelles. Furthermore, we have shown that the effects on one end of the protein can be relayed to long distance sites, providing important information that can aid in protein engineering efforts.