The closed conformation of a highly flexible protein: The structure of E. coli adenylate kinase with bound AMP and AMPPNP

Abstract

The structure of E. coli adenylate kinase with bound AMP and AMPPNP at 2.0 A resolution is presented. The protein crystallizes in space group C2 with two molecules in the asymmetric unit, and has been refined to an R factor of 20.1% and an Rfree of 31.6%. In the present structure, the protein is in the closed (globular) form with the large flexible lid domain covering the AMPPNP molecule. Within the protein, AMP and AMPPNP, and ATP analog, occupy the AMP and ATP sites respectively, which had been suggested by the most recent crystal structure of E. coli adenylate kinase with Ap5A bound (Müller and Schulz, 1992, ref. 1) and prior fluorescence studies (Liang et al., 1991, ref. 2). The binding of substrates and the positions of the active site residues are compared between the present structure and the E. coli adenylate kinase/Ap5A structure. We failed to detect a peak in the density map corresponding to the Mg2+ ion which is required for catalysis, and its absence has been attributed to the use of ammonium sulfate in the crystallization solution. Finally, a comparison is made between the present structure and the structure of the heavy chain of muscle myosin.

Illuminating the mechanistic roles of enzyme conformational dynamics

“…2b, demonstrates that the lid domain of AK is in dynamical equilibrium between the open and closed conformational modes with each mode containing an ensemble of substates. Our results are in sharp contrast to the common view that AK's lid should remain open in the absence of substrates (13,(15)(16)(17), whereas binding would cause a global conformational change resulting in lid closure. That the substrate-free enzyme should remain in the open form has also been assumed (a reasonable supposition given the existing ensemble-averaged results) in the analysis of NMR relaxationdispersion experiments performed on AK (19).…”

“…Rather, interaction with substrates changes the enzyme's conformational dynamics, doubling the lid closing rate upon ligand binding. The increase in the closing rate is likely driven by electrostatic interaction between positively charged residues in AK's binding pocket and the negatively charged phosphate groups on the ligand (13). Perhaps more interestingly, the opening rates of AK's lid domain are comparable within experimental errors.…”

“…AK was chosen for this study because many aspects of this enzyme have been studied both experimentally (11)(12)(13)(14)(15)(16)(17)(18)(19) and theoretically (20)(21)(22)(23)(24). It is an ubiquitous enzyme that helps maintain the energy balance in cells by catalyzing the reversible reaction, Mg 2ϩ ⅐ATP ϩ AMP º Mg 2ϩ ⅐ADP ϩ ADP.…”

Domain Flexibility in Ligand-Free and Inhibitor-BoundEscherichia coliAdenylate Kinase Based on a Mode-Coupling Analysis of15N Spin Relaxation

“…These studies also indicated that large-amplitude segmental mobility of AMPbd and LID is in effect in the ligand-free form (23). AKeco is the only adenylate kinase for which crystal structures corresponding to the extreme stages of the catalytic cycle are available (17,21,22), with the closed form represented by AKeco*AP 5 A. The ATP phosphates are bound to the enzyme partly through the so-called P-loop GXXGXGK (residues [7][8][9][10][11][12][13].…”

An automatic method involving cluster analysis of secondary structures for the identification of domains in proteins

“…The three clusters in the secondary structural dendrogram of guanylate kinase (1GKY) correspond to these two domains and a cluster of two roughly antiparallel helices. The third cluster corresponds to a highly flexible "flap" or "lid" domain in the adenylate kinases (1AKE and 1AK3) that undergoes large movements upon AMP binding (Berry et al, 1994) not present in the guanylate kinases. Indeed, the secondary structural dendrograms of adenylate kinases have three clusters representing the three domains.…”