During assembly of many viruses a powerful ATP-driven motor translocates DNA into a preformed procapsid. A Walker-A “P-loop” motif is proposed to coordinate ATP binding and hydrolysis with DNA translocation. We use genetic, biochemical, and biophysical techniques to survey the roles of P-loop residues in bacteriophage lambda motor function. We identify 55 point mutations that reduce virus yield to below detectible levels in a highly sensitive genetic complementation assay and 33 that cause varying reductions in yield. Most changes in the predicted conserved residues K76, R79, G81, and S83 produce no detectible yield. Biochemical analyses show that R79A and S83A mutant proteins fold, assemble, and display genome maturation activity similar to wild-type, but exhibit little ATPase or DNA packaging activity. Kinetic DNA cleavage and ATPase measurements implicate R79 in motor ring assembly on DNA, supporting recent structural models that locate the P-loop at the interface between motor subunits. Single-molecule measurements detect no translocation for K76A&R, while G81A and S83A exhibit strong impairments consistent with their predicted roles in ATP binding. We identify eight residue changes spanning A78-K84 that yield impaired translocation phenotypes and show that Walker-A residues play important roles in determining motor velocity, pausing, and processivity. The efficiency of initiation of packaging correlates strongly with motor velocity. Frequent pausing and slipping caused by changes A78V and R79K suggest these residues are important for ATP alignment and coupling of ATP binding to DNA gripping. Our findings support recent structural models implicating the P-loop arginine in ATP hydrolysis and mechanochemical coupling.
Background: Stimulation by post-hydrolytic, ADP-bound conformations of SUR1 underlies current models of K ATP channel activation; ATP analogs are assumed to lower activity by reducing hydrolysis. Results: ATP␥S switches conformations with lowered affinity; AMP-PxP selectively bind NBD1, preventing switching.
Conclusion:The actions of ATP analogs on K ATP channels require reinterpretation. Significance: Reduced affinities of SUR1 NBDs for ATP analogs limit conformational switching and channel activity.
The clearance of retinoic acid (RA) and its metabolites is believed to be regulated by the CYP26 enzymes, but the specific roles of CYP26A1, CYP26B1, and CYP26C1 in clearing active vitamin A metabolites have not been defined. The goal of this study was to establish the substrate specificity of CYP26C1, and determine whether CYP26C1 interacts with cellular retinoic acid binding proteins (CRABPs). CYP26C1 was found to effectively metabolize all- retinoic acid (RA), 9--retinoic acid (9--RA), 13--retinoic acid, and 4-oxo-RA with the highest intrinsic clearance toward 9--RA. In comparison with CYP26A1 and CYP26B1, CYP26C1 resulted in a different metabolite profile for retinoids, suggesting differences in the active-site structure of CYP26C1 compared with other CYP26s. Homology modeling of CYP26C1 suggested that this is attributable to the distinct binding orientation of retinoids within the CYP26C1 active site. In comparison with other CYP26 family members, CYP26C1 was up to 10-fold more efficient in clearing 4-oxo-RA (intrinsic clearance 153 l/min/pmol) than CYP26A1 and CYP26B1, suggesting that CYP26C1 may be important in clearing this active retinoid. In support of this, CRABPs delivered 4-oxo-RA and RA for metabolism by CYP26C1. Despite the tight binding of 4-oxo-RA and RA with CRABPs, the apparent Michaelis-Menten constant in biological matrix () value of these substrates with CYP26C1 was not increased when the substrates were bound with CRABPs, in contrast to what is predicted by free drug hypothesis. Together these findings suggest that CYP26C1 is a 4-oxo-RA hydroxylase and may be important in regulating the concentrations of this active retinoid in human tissues.
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