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More than 30 mutations in ACTA2, which encodes α-smooth muscle actin, have been identified to cause autosomal dominant thoracic aortic aneurysm and dissection. The mutation R256H is of particular interest because it also causes patent ductus arteriosus and moyamoya disease. R256H is one of the more prevalent mutations and, based on its molecular location near the strand-strand interface in the actin filament, may affect F-actin stability. To understand the molecular ramifications of the R256H mutation, we generated Saccharomyces cerevisiae yeast cells expressing only R256H yeast actin as a model system. These cells displayed abnormal cytoskeletal morphology and increased sensitivity to latrunculin A. After cable disassembly induced by transient exposure to latrunculin A, mutant cells were delayed in reestablishing the actin cytoskeleton. In vitro, mutant actin exhibited a higher than normal critical concentration and a delayed nucleation. Consequently, we investigated regulation of mutant actin by formin, a potent facilitator of nucleation and a protein needed for normal vascular smooth muscle cell development. Mutant actin polymerization was inhibited by the FH1-FH2 fragment of the yeast formin, Bni1. This fragment strongly capped the filament rather than facilitating polymerization. Interestingly, phalloidin or the presence of wild type actin reversed the strong capping behavior of Bni1. Together, the data suggest that the R256H actin mutation alters filament conformation resulting in filament instability and misregulation by formin. These biochemical effects may contribute to abnormal histology identified in diseased arterial samples from affected patients.

Intramolecular allosteric interactions responsible for actin conformational regulation are largely unknown. Previous work demonstrated that replacing yeast actin Val-76 with muscle actin Ile caused decreased nucleotide exchange. Residue 76 abuts Trp-79 in a six-residue linear array beginning with Lys-118 on the surface and ending with His-73 in the nucleotide cleft. To test if altering the degree of packing of these two residues would affect actin dynamics, we constructed V76I, W79F, and W79Y single mutants as well as the Ile-76/Phe-79 and Ile-76/Tyr-79 double mutants. Tyr or Phe should decrease crowding and increase protein flexibility. Subsequent introduction of Ile should restore packing and dampen changes. All mutants showed decreased growth in liquid medium. W79Y alone was severely osmosensitive and exhibited vacuole abnormalities. Both properties were rescued by Ile-76. Phe-79 or Tyr decreased the thermostability of actin and increased its nucleotide exchange rate. These effects, generally greater for Tyr than for Phe, were reversed by introduction of Ile-76. HD exchange showed that the mutations caused propagated conformational changes to all four subdomains. Based on results from phosphate release and light-scattering assays, single mutations affected polymerization in the order of Ile, Phe, and Tyr from least to most. Introduction of Ile-76 partially rescued the polymerization defects caused by either Tyr-79 or Phe-79. Thus, alterations in crowding of the 76–79 residue pair can strongly affect actin conformation and behavior, and these results support the theory that the amino acid array in which they are located may play a central role in actin regulation.

Muscle and yeast actins display distinct behavioral characteristics. To better understand the allosteric interactions that regulate actin function, we created a muscle/yeast hybrid actin containing a muscle-specific outer domain (subdomains 1 and 2) and a yeast inner domain (subdomains 3 and 4). Actin with muscle subdomain 1 and the two yeast N-terminal negative charges supported viability. The four negative charge muscle N terminus in a muscle subdomain 1 background caused death, but in the same background actin with three N-terminal acidic residues (3Ac/Sub1) led to sick but viable cells. Addition of three muscle subdomain 2 residues (3Ac/Sub12) produced no further deleterious effects. These hybrid actins caused depolarized cytoskeletons, abnormal vacuoles, and mitochondrial and endocytosis defects. 3Ac/Sub1 G-actin exchanged bound epsilonATP more slowly than wild type actin, and the exchange rate for 3Ac/Sub12 was even slower, similar to that for muscle actin. The mutant actins polymerized faster and produced less stable and shorter filaments than yeast actin, the opposite of that expected for muscle actin. Unlike wild type actin, in the absence of unbound ATP, polymerization led to ADP-F-actin, which rapidly depolymerized. Like yeast actin, the hybrid actins activated muscle myosin S1 ATPase activity only about one-eighth as well as muscle actin, despite having essentially a muscle actin-specific myosin-binding site. Finally, the hybrid actins behaved abnormally in a yeast Arp2/3-dependent polymerization assay. Our results demonstrate a unique sensitivity of yeast to actin N-terminal negative charge density. They also provide insight into the role of each domain in the control of the various functions of actin.