The magnesium-ATP-binding site on chicken gizzard myosin light chain kinase remains open and functionally competent during the calmodulin-dependent activation-inactivation cycle of the enzyme

Abstract

An ATP-like affinity labeling reagent, 5'-[p-(fluorosulfonyl)benzoyl]adenosine (FSBA), was used to probe the MgATP-binding site of smooth muscle myosin light chain kinase from chicken gizzard (smMLCK) and its calmodulin (CaM) complex. Native smMLCK has an absolute requirement for the binding of the calcium complex of CaM for expression of its catalytic activity. FSBA reacted with smMLCK-CaM and with the CaM-free, inactive enzyme as well. Both reactions were dependent on time and FSBA concentration. Reaction was accompanied by the incorporation of covalently bound [14C]FSBA into smMLCK protein at a molar ratio of approximately 1:1 in each case. p-(Fluorosulfonyl)benzoic acid, an analogue of FSBA lacking the adenosine targeting group, did not react at a significant rate with either form of smMLCK. Reaction of CaM-free and CaM-bound smMLCK with FSBA displayed saturation kinetics. The first-order rate constants for the conversion of the reversible, noncovalent enzyme-FSBA complex to form the irreversibly inhibited, covalently modified enzyme were similar for both smMLCK and smMLCK-CaM, 0.15 and 0.07 min-1, respectively. The concentrations of FSBA yielding the half-maximal rate of inactivation, KI, were essentially identical--0.65 and 0.64 mM, respectively--for smMLCK and smMLCK-CaM. MgATP, but not MgGTP or a substrate peptide, potently inhibited reaction with FSBA. Inhibition by MgATP was competitive. The measured inhibitory constant for MgATP was essentially the same--33 versus 34 microM--for both smMLCK and smMLCK-CaM. It therefore is concluded that the MgATP-binding site on smMLCK remains accessible and recognizable as such when the enzyme becomes inactivated upon dissociation of CaM.

Affinity labelling of smooth-muscle myosin light-chain kinase with 5′-[p-(fluorosulphonyl)benzoyl]adenosine

“…This suggests that the ATP-binding site is open to the substrate, regardless of calmodulin binding. Similar results have been obtained for skeletal-muscle MLCK (Colburn et al, 1987) and recently for smooth-muscle MLCK (Kennelly et al, 1992).…”

Synapsins as major neuronal Ca2+/S100A1-interacting proteins

“…This is conceivable in view of the synapsin crystal structure in which the nucleotide binding site is located in a wide open pocket that does not have direct contacts with the S100A1\ calmodulin-binding site [22]. However, protein kinases such as smooth-muscle or skeletal-muscle myosin light-chain kinase, which require Ca# + \calmodulin for activity, can similarly be labelled with FSBA in the absence of Ca# + \calmodulin [31,32]. Therefore the fact that the FSBA labelling is independent of Ca# + \S100A1 does not exclude the possibility that S100A1 could have some role in the regulation of the currently unknown catalytic activity of the synapsins.…”

Structure and Regulation of Calcium/Calmodulin-Dependent Protein Kinases

“…The smaller N-terminal domain of the catalytic core of MLCK binds MgATP under a glycine-rich flap in the presence or absence of Ca 2+ /CaM, indicating that the ATP-binding pocket is exposed and competent during autoinhibition; however, the protein substrate, RLC, does not bind . The orientation of the N-terminus of RLC in the cleft may be similar to the orientation of the substrate recognition fragment of the inhibitor peptide of cAMP-dependent protein kinase .…”