title: A potential yeast actin allosteric conduit dependent on hydrophobic core residues val-76 and trp-79.
abstract: 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.
authors.affiliation: Department of Biochemistry, University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, IA 52242, USA.
authors.forename: Kuo-Kuang
authors.initials: KK
authors.lastname: Wen
authors.forename: Melissa
authors.initials: M
authors.lastname: McKane
authors.forename: Ema
authors.initials: E
authors.lastname: Stokasimov
authors.forename: Jonathon
authors.initials: J
authors.lastname: Fields
authors.forename: Peter A
authors.initials: PA
authors.lastname: Rubenstein
mesh_terms: D000199:Actins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D000494:Allosteric Regulation; D019943:Amino Acid Substitution; D003470:Culture Media; D003599:Cytoskeleton / Q000648:ultrastructure; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics; D008958:Models, Molecular; D016296:Mutagenesis; D010957:Plasmids; D011487:Protein Conformation; D014364:Tryptophan / Q000032:analysis; D014617:Vacuoles / Q000648:ultrastructure; D014633:Valine / Q000032:analysis; D015003:Yeasts / Q000235:genetics* / Q000254:growth & development
chemical_list: D000199:Actins; D003470:Culture Media; D005656:Fungal Proteins; D014364:Tryptophan; D014633:Valine