EFFECT OF METAL-CATIONS ON THE CONFORMATION OF MYOSIN SUBFRAGMENT-1-ADP-PHOSPHATE ANALOG COMPLEXES - A NEAR-UV CIRCULAR-DICHROISM STUDY

The interaction of myosin with actin, coupled with hydrolysis of ATP, is the molecular basis of muscle contraction. The head segment of myos in, called S1, contains the distinct binding sites for ATP and actin a nd is responsible for the ATPase activity. The myosin-catalyzed ATP hy drolysis consists of several intermediate steps and each step is accom panied by conformational changes in the S1 segment. The rate-limiting step of the ATP hydrolysis is the dissociation of the S1 . ADP . P-i c omplex which is accelerated by actin. The substitution of P-i with pho sphate analogs (PA), such as vanadate, beryllium fluoride (BeFx) or al uminum fluoride (AIF(4)(-)), yields stable complexes which mimic the i ntermediates of the ATP hydrolysis. In this work, tertiary structure c hanges in S1 in the vicinity of aromatic residues was studied by compa ring near-UV circular dichroism (CD) spectra from S1-nucleotide-phssph ate analog complexes in the presence of Mg2+ and other cations. A sign ificant difference between the MgATP and MgADP spectra indicated notab le tertiary structural changes accompanying the M*ADP . P-i --> M*ADP transition. The spectra of the S1 . MSADP . BeFx and S1 . MgADP . AlF 4- complexes resemble to those obtained upon addition of MgATP gamma S and MgATP to S1, and correspond to the M. ATP and M**. ADP . P-i int ermediates, respectively. We have found recently that the presence of divalent metal cations (Me2+) is essential for the formation of stable S1 . MeADP . PA complexes. Moreover, the nature of the metal cations strongly influences the stability of these complexes [Peyser, Y. M., e t al. (1996) Biochemistry 35, 4409-4416]. In the present work we studi ed the effect of Mg2+, Mn2+, Ca2+, Ni2+, Co2+, and Fe2+ on the near-UV CD spectrum of the ATP, ADP, ADP . BeFx, and ADP . AlF4- containing S 1 complexes. The CD spectra obtained with ADP, ATP ADP . BeFx and ADP . AlF4- were essentially identical in the presence of Co2+ and rather similar in the case of Ca2+, while they were partially different in ot her cases. An interesting correlation was found between actin activati on and ATP versus ADP difference spectra in the presence of various me tal ions. The distribution of the fractional concentration of the inte rmediates of ATP hydrolysis was estimated in the presence of each cati on from the CD spectra with phosphate analogs. In the presence of Mg2 the predominant intermediate is the M*. ADP . P-i state, which is in accordance with the kinetic studies. On the other hand with non-nativ e cations the predominant intermediate is the M. ADP state and the re lease of ADP is the rate limiting step in the myosin-catalyzed ATP hyd rolysis. According to the results, the near-UV CD spectrum originating from aromatic residues in S1 not only can distinguish identifiable st ates in the ATP hydrolysis cycle but can also pinpoint to changes in t he tertiary structure caused by complex formation with nucleotide or n ucleotide analog and various divalent metal cations. These findings, t hat are correlative with actin activation, and thus with the power str oke, suggest new strategies for perturbing S1 structure in the continu ous efforts directed toward the elucidation of the mechanism of muscle contraction.