STRUCTURE OF THE BETA(2) HOMODIMER OF BACTERIAL LUCIFERASE FROM VIBRIO-HARVEYI - X-RAY-ANALYSIS OF A KINETIC PROTEIN-FOLDING TRAP

Luciferase, as isolated from Vibrio harveyi, is an alpha beta heterodi mer. When allowed to fold in the absence of the alpha subunit, either in vitro or in vivo, the beta subunit of the enzyme will form a kineti cally stable homodimer that does not unfold even after prolonged incub ation in 5 M urea at pH 7.0 and 18 degrees C. This form of the beta su bunit, arising via kinetic partitioning on the folding pathway, appear s to constitute a kinetically trapped alternative to the heterodimeric enzyme (Sinclair JF, Ziegler MM, Baldwin TO. 1994. Kinetic partitioni ng during protein folding yields multiple native states. Nature Struct Biol 1: 320-326). Here we describe the X-ray crystal structure of the beta(2) homodimer of luciferase from V. harveyi determined and refine d at 1.95 Angstrom, resolution. Crystals employed in the investigation belonged to the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions of a = 58.8 Angstrom, b = 62.0 Angstrom, and c = 218.2 Ang strom and contained one dimer per asymmetric unit. Like that observed in the functional luciferase alpha beta heterodimer, the major tertiar y structural motif of each beta subunit consists of an (alpha/beta)(8) barrel (Fisher AJ, Raushel FM, Baldwin TO, Rayment I. 1995. Three-dim ensional structure of bacterial luciferase from Vibrio harveyi at 2.4 Angstrom resolution. Biochemistry 34: 6581-6586). The root-mean-square deviation of the alpha-carbon coordinates between the beta subunits o f the hetero- and homodimers is 0.7 Angstrom. This high resolution X-r ay analysis demonstrates that ''domain'' or ''loop'' swapping has not occurred upon formation of the beta(2) homodimer and thus the stabilit y of the beta(2) species to denaturation cannot be explained in such s imple terms. In fact, the subunit:subunit interfaces observed in both the beta(2) homodimer and alpha beta heterodimer are remarkably simila r in hydrogen-bonding patterns and buried surface areas.