Nucleotide and Polymerization Effects on Actin Structure and Dynamics
Jepsen, Lauren
2018
Abstract
Actin is one of the most highly conserved and abundant proteins found in eukaryotic cells, essential for determining cell shape, polarity and motility, as well as protein transport and even mitosis and muscle contraction. These functions depend on actin’s ability to exist – under the strict control of nucleotide hydrolysis and interaction with actin binding partners – as either a monomer (G-actin) or a polymer (F-actin). Over the last seven decades of research, much has been determined in the way of actin function, but key questions still remain. First, the affinity of actin for the >150 actin binding proteins, and for polymerization, is reliant on upon actin’s nucleotide state but the structural changes that occur upon nucleotide hydrolysis are not so clear. Next, the two ends of the actin filament have different properties, with incoming ATP-actin subunits preferentially adding to the barbed end of the filament and ADP-actin subunits preferentially dissociating from the pointed end. Much like with the structural changes that occur upon hydrolysis, the physical and biochemical bases for these differences are unknown. Finally, as the cell relies on the strict regulation of the actin filament, alterations to actins sequence are poorly tolerated. Although many mutations prove to be lethal to the organism, over 140 disease causing actin mutants have been reported, with a large subset clustering on actin’s pathogenic helix (residues 113-125). Little is known about the structural consequences of these mutations and how they relate to disease. In my thesis, I use molecular dynamics simulations of both G- and F-actin to probe these questions. Actin itself has two clefts, the nucleotide-binding cleft at the center of the protein, and the target-binding cleft at the bottom of the protein between subdomains 1 and 3, where the majority of actin binding proteins dock. I show that changes within the nucleotide-binding cleft propagate down to the target-binding cleft through the intermediary C-terminal hinge (A331-Y337). Within the target-binding cleft itself, I identify a new loop at the profilin binding site (FQQ-loop: S348-W356) that moves by nearly 5 Å in the ATP state to partially obstruct the target-binding cleft. All of these changes help explain nucleotide state specificity for actin binding proteins. My work also reveals that ATP G-actin takes on a flatter conformation that is structurally similar to F-actin’s barbed end protomer, explaining the observation that ATP G-actin polymerizes faster than its ADP counterpart. I find that the pointed end of the filament takes on a conformation that is divergent from remainder of the filament and monomer simulations, effectively raising the conformational energy barrier for the addition of actin protomers. I also looked at the structural consequences of the deafness causing mutations K118M/N. The mutations to K118 result in changes in the structure and dynamics of the D-loop, alterations in the structure of the H73-loop as well as the sidechain orientations of W79 and W86, changes in nucleotide exchange rates, and significant shifts in the twist of the actin monomer. With K118N the twist of the monomer is nearly identical to the F-actin protomer, and in vitro polymerization assays show that this mutation results in faster polymerization. Taken together, it is evident that mutations at this site give rise to a series of small changes that can be tolerated in vivo, but result in misregulation of actin assembly and dynamics.Subjects
Actin Molecular Dynamics DFNA20/26 Polymerization
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