Dual Roles of Gln137 of Actin Revealed by Recombinant Human Cardiac Muscle α-Actin Mutants

Abstract

The actin filament is quite dynamic in the cell. To determine the relationship between the structure and the dynamic properties of the actin filament, experiments using actin mutants are indispensable. We focused on Gln 137 to understand the relationships between two activities: the conformational changes relevant to the G-to F-actin transition and the activation of actin ATPase upon actin polymerization. To elucidate the function of Gln 137 in these activities, we characterized Gln 137 mutants of human cardiac muscle ␣-actin. Although all of the single mutants, Q137E, Q137K, Q137P, and Q137A, as well as the wild type were expressed by a baculovirus-based system, only Q137A and the wild type were purified to high homogeneity. The CD spectrum of Q137A was similar to that of the wild type, and Q137A showed the typical morphology of negatively stained Q137A F-actin images. However, Q137A had an extremely low critical concentration for polymerization. Furthermore, we found that Q137A polymerized 4-fold faster, cleaved the ␥-phosphate group of bound ATP 4-fold slower, and depolymerized 5-fold slower, as compared with the wild-type rates. These results suggest that Gln 137 plays dual roles in actin polymerization, in both the conformational transition of the actin molecule and the mechanism of ATP hydrolysis.

Filament Structure, Organization, and Dynamics in MreB Sheets

“…The kinetic dynamics of Thermatoga MreB-NTP filament formation was faster than F-actin but significantly slower than ParM (8,21). The critical concentration of MreB-NTP filament formation was similar to ParM, yet assembly under conditions closer to physiological occurred without a nucleation step, making Thermatoga MreB a very efficient polymerizing machine.…”

Role of the Actin Ala-108–Pro-112 Loop in Actin Polymerization and ATPase Activities

“…The N-terminal amino acid sequence of the WT actin is MGWSHPQFEKGGIEGRDDEE; the underlined part is an additional amino acid sequence including the Strep-tag II for purification. The extra N-terminal sequence did not affect the polymerization path of the WT actin, although it modified the rate constant, and furthermore, it hardly affected the overall actin ATPase activity (8). The transfer vector was pVL1392-L21, including the enhancer sequence L21 in the pVL1392 vector (Pharmingen) (11).…”

“…Recombinant human cardiac muscle wild type ␣-actin (the WT actin) was prepared according to the method we described previously (8). The N-terminal amino acid sequence of the WT actin is MGWSHPQFEKGGIEGRDDEE; the underlined part is an additional amino acid sequence including the Strep-tag II for purification.…”

“…The loop shifts substantially relative to subdomain 3 upon sliding. Moreover, the preceding part of the loop contacts the side chain of Gln-137, which participates in both the conformational transition of the actin molecule and the ATPase reaction (8,9). The successive part of the loop contacts the diagonal subunit in F-actin.…”

Nuclear actin-related proteins take shape

“…Together with the exchange of catalytically important residues (His 161 and Gln 137 of actin are exchanged by Ser 166 and Thr 142 in Arp4 respectively) this might lead to a stable ATP bound state of the protein. [22][23][24] This does not rule out the possibility that Arp4's ATPase activity might be triggered in the cellular context, e.g., by binding to other interaction partners. On the other hand, ATP might simply be a structural component to stabilize the fold of Arp4.…”