X-ray crystallographic analysis of the pyrophosphate-dependent phosphofructokinase of Spirochaeta thermophilum : a thesis presented in partial fulfillment of the requirements for the degree of Master of Science in Biochemistry at Massey University, New Zealand
The structure of a homodimeric, non-allosteric, PPᵢ.-dependent phosphofructokinase from the thermophilic bacterium Spirochaeta thermophilum has been resolved by X-ray crystallography in two distinct conformations at 2.2 (R = 0.1991 [R
= 0.2288]) and 1.85 Å (R = 0.1923 [R
= 0.2035]) resolution. The 554 residue (Mᵣ 61080 g.mol-¹) subunit, a homologue of the plant PPᵢ,-PFK β-subunit exhibits an asymmetrical quaternary structure and shares both sequence and tertiary structure with the N- and C- terminal Rossmann-like domains of prokaryotic ATP-PFKs. Spirochaeta thermophilum PPᵢ-PFK exhibits three major inserts relative to the prokaryotic ATP-PFK of E. coli, an N-terminal insert, a C-terminal insert, and an insert within the PFK C-terminal domain which forms an autonomous α-helical domain. The active site is formed at the interface of the N and C domains. The 'open' and 'closed' subunit asymmetry of the S. thermophilum PPᵢ-PFK 1.85 Å atomic model mirrors that of the B. burgdorferi PPᵢ-PFK (1KZH [Moore et al.2002]) with the exception that the two unique β-hairpins (380-390 [α16-α17] and 485-495 [β14-β15]) of subunit A are not displaced into the active site. Both subunits of the S. thermophilum PPᵢ-PFK 2.2 Å atomic model adopt an 'open', apparently inactive conformation. The conformational change involves concomitant closure of the active site of both subunits via a rigid-body displacement of the C and α- helical domains, relative to the N domain. The N domain of one subunit and the C domain of the opposing subunit can be thought of as a rigid body, therefore closure of one active site dictates closure of the other. Rotation of the small domain forces Met251 of the MGR motif to adopt an active conformation and displacement of the α-helical domain, specifically the 380-390 β-hairpin into the active site 'folds' Arg253 (MGR) into an active conformation. Closure of the active site, which prevents wasteful hydrolysis, involves movement of the β14-β15 β-hairpin into the active site and simultaneous rearrangement of the PPᵢ-binding GGDD motif. The conformational change of the S. thermophilum PPᵢ-PFK is surprisingly complex and unique relative to prokaryotic ATP-PFKs and involves displacement of novel structural elements. These movements change the conformation of conserved motifs at the active site and therefore function to modulate PPᵢ-dependent activity.