The role of phosphagen specificity loops in arginine kinase

Abstract

Phosphagen kinases catalyze the reversible transfer of a phosphate between ATP and guanidino substrates, a reaction that is central to cellular energy homeostasis. Members of this conserved family include creatine and arginine kinases and have similar reaction mechanisms, but they have distinct specificities for different guanidino substrates. There has not been a full structural rationalization of specificity, but two loops have been implicated repeatedly. A small domain loop is of length that complements the size of the guanidino substrate, and is located where it could mediate a lock-and-key mechanism. The second loop contacts the substrate with a valine in the methyl-substituted guanidinium of creatine, and with a glutamate in the unsubstituted arginine substrate, leading to the proposal of a discriminating hydrophobic/hydrophilic minipocket. In the present work, chimeric mutants were constructed with creatine kinase loop elements inserted into arginine kinase. Contrary to the prior rationalizations of specificity, most had measurable arginine kinase activity but no creatine kinase activity or enhanced phosphocreatine binding. Guided by structure, additional mutations were introduced in each loop, recovering arginine kinase activities as high as 15% and 64% of wild type, respectively, even though little activity would be expected in the constructs if the implicated sites had dominant roles in specificity. An atomic structure of the mismatched complex of arginine kinase with creatine and ADP indicates that specificity can also be mediated by an active site that allows substrate prealignment that is optimal for reactivity only with cognate substrates and not with close homologs that bind but do not react.

Figure 1.

Chemical structure of representative phosphagens.

Figure 2.

Transition state analog structures of arginine kinase (red [Zhou et al. 1998; Yousef et al. 2002]) and creatine kinase (blue [Lahiri et al. 2002]) superimposed with protein drawn as ribbon and substrates as stick-model, showing the “specificity loops” of each domain.

Figure 3.

The phosphagen binding site. (A) Creatine bound in the active site of Limulus arginine kinase. The electron density shown in stereo is of a F_o−F_c sigma-A weighted difference electron density map, contoured at 1.9 σ. The map was calculated before creatine had been added to the model. Although maps later in the refinement were of higher quality, this one has had no opportunity for the introduction of phase bias, so the distinctive protrusion for the creatine methyl group is confirmation of the contents of the active site. For clarity, only a selection of residues from the neighboring large and small domain specificity loops is shown. (B) Interactions of arginine with arginine kinase in its transition state analog complex (red [Zhou et al. 1998; Yousef et al. 2002]), for comparison with the creatine interactions shown in panels A and C. Probable hydrogen bonds are shown with dotted lines. (C) The arginine kinase active site with structures of the arginine and creatine transition state like complexes superimposed. The arginine complex is shown in purple, the creatine complex in green. The creatine complex was crystallized with ATP but no nitrate. Thus, after apparent hydrolysis in the crystal, there is nothing occupying the site of the γ-phosphate. The most noticeable difference between the two structures is rotation of the substrate guanidinium by ~30 degrees. (D) Comparison of the arginine kinase-creatine-ADP active site (orange carbons) with the creatine kinase transition state analog structure (green carbons [Lahiri et al. 2002]). Again, the main difference is rotation of the guanidinium group. Arginine kinase residues are labeled with parentheses, creatine kinase, without. (E) Location of the E59-K16 salt bridge with respect to the active site. The transition state analog structure (Zhou et al. 1998; Yousef et al. 2002) is shown in ribbon form. In ball-and-stick form are the substrate analog atoms, and residues 16 and 59, which form the salt bridge.