Functional roles of ionic and hydrophobic surface loops in smooth muscle myosin: their interactions with actin.
This investigation ascertains whether, in (smooth muscle) myosin, certain residues engage in functional interactions with their actin conjugates in an actomyosin complex. Such interactions have been postulated from putting together crystallographic models of the two proteins [Rayment, I., Rypniewski, W. R., Schmidt-Bäse, K., Smith, R., Tomchick, D. R., Benning, M. M., Winkelmann, D. A., Wesenberg, G., and Holden, H. M. (1993) Science 261, 50-58]. Here, in several instances, we ask whether mutation of a particular residue significantly impairs a function, and find that the answers are largely rationalized by the original postulation. Additionally, a novel element emerges from our investigation. To assess function, we test the wild type and mutant systems as they perform in the steady state of ATP degradation. In doing so, we assume, as usual, that degradation proceeds from an early stage in which the complex forms (and is described by parameter K(app)) to a later stage during which the product leaves the complex (and is described by parameter V(max)). Interestingly, certain defects induced by the mutations are associated with changes in K(app), and other defects are associated with changes in V(max), suggesting that our procedure at least roughly distinguishes between events according to the time in the degradation at which they occur. In this framework, we suggest that (1) in the actin-myosin association phase, cationic residues Lys-576 and Lys-578 interact with anionic residues of the so-called second actin, and (2) in the product leaving phase, hydrophobic residues Trp-546, Phe-547, and Pro-548, as well as the Thr-532/Asn-533/Pro-534/Pro-535 sequence, sever connections with the so-called first actin. The role of Glu-473 is also examined.
Kojima
S
S
Department of Structural Analysis, National Cardiovascular Center Research Institute, Fujishiro-dai, Suita, Osaka 565-8565, Japan.
Konishi
K
K
Katoh
K
K
Fujiwara
K
K
Martinez
H M
HM
Morales
M F
MF
Onishi
H
H
D000199:Actins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology* / Q000648:ultrastructure; D000818:Animals; D002417:Cattle; D004789:Enzyme Activation / Q000235:genetics; D046911:Macromolecular Substances; D009130:Muscle, Smooth / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D015879:Myosin Subfragments / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000648:ultrastructure; D009218:Myosins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000502:physiology* / Q000648:ultrastructure; D017433:Protein Structure, Secondary; D011817:Rabbits; D013499:Surface Properties
D000199:Actins; D046911:Macromolecular Substances; D015879:Myosin Subfragments; D009218:Myosins