Advisory Committee Chair
Kevin L Kirk
Advisory Committee Members
David M Bedwell
Narayana Vl Sthanam
Robin Aj Lester
James C Patterson
John L Hartman, Iv
Document Type
Dissertation
Date of Award
2017
Degree Name by School
Doctor of Philosophy (PhD) College of Arts and Sciences
Abstract
The concerted model of allosteric protein regulation suggests that the conformational states of a protein are tightly coupled. Therefore, modifications to a protein (or its substrate) that biases the system towards a specific state will also affect the structure and function of other protein domains, even if the site of the modification and the affected domains do not appear to directly interact. These modfications can manifest as gain-of-function (e.g., biasing the active state and thereby promoting protein function and increasing the affinity for activating substrates), loss-of-function (e.g., biasing the inactive state and thereby antagonizing protein function and decreasing the affinity for activating substrates), or otherwise alter the free energy profile along a protein's reaction coordinate. These free energy differences can be assessed biochemically by measuring protein function and substrate affinity or computationally/structurally by determining the frequency of time that a protein spends in certain conformations along its reaction coordinate. In this work we identified several mutations to the cystic fibrosis transmembrane regulator (CFTR) that bias the CFTR ion channel to its open-state, thereby promoting channel opening, enhancing ATP (and other agonist) sensitivity, and rescuing secondary loss-of-function defects. The mutations were located in varied CFTR domains (e.g., an extracellular gating residue, an intracellular loop that couples the nucleotide-binding domains to the transmembrane-spanning domains, a pore-lining transmembrane helix, and a putative intraceullular gate) and yet all exerted effects on regions of the protein that did not directly contact the mutation sites. Remarkably, a subset of the analogous mutations in other multidrug resistance proteins (of which CFTR is a member) also resulted in gain-of-function effects. This is notable since the MRPs are active ATPase pumps, not an ATP-gated channel like CFTR, indicating that the MRPs and CFTR may share a conserved allosteric network despite their thermodynamic differences. We have also addressed the impact of modifications to the principal substrate (a conserved sorting signal sequence) of the bacterial Sortase A (SrtA) protein via atomistic free molecular dynamics simulations. This work was performed in order to help clarify conflicting empirical structures of the SrtA-sorting signal complex. Like the MRPs and CFTR, we show that the structure and function of various SrtA domains are influenced by the addition of a carboxybenzyl protecting group to the sorting signal peptide. These changes manifest as alterations in the structural ensemble clusters and essential dynamics of the protein generated from the molecular dynamics simulation trajectories. Here too we can see the indirect effects of a modified substrate as the substrate itself appears to couple distinct regions of the SrtA protein in a cross-correlation analysis, although additional studies will need to be performed on the SrtA apo configuration to confirm this hypothesis. Taken together, this work demonstrates several examples of indirect effects on protein function stemming from site-directed mutations and modified substrates, in thermodynamically-distinct proteins.
Recommended Citation
Roessler, Bryan C., "Indirect Interactions Affect the Dynamics of Multidrug Resistance Proteins and Sortase A" (2017). All ETDs from UAB. 2852.
https://digitalcommons.library.uab.edu/etd-collection/2852
