Subunit conformation and dynamics in a heterodimeric protein: studies of the hybrid isozyme of creatine kinase

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Abstract

Several physical properties of creatine kinase (EC 2.7.3.2) isozymes MM (CK-MM, muscle-type) and BB (CK-BB, brain-type), both homodimers, and isozyme MB (CK-MB), a heterodimer, were compared to determine how formation of the hybrid modifies subunit conformation and dynamics. Circular dichroic spectra revealed additional α-helical content for the hybrid isozyme. Double-beam absorption difference spectra between CK-MB and a stoichiometric mixture of CK-MM and CK-BB revealed decreased exposure of intrinsic chromophores in the hybrid. The relative intensity of the intrinsic fluorescence of CK-MB was between the two homodimers, but was 16% closer to the less fluorescent CK-MM. Steady state anisotropy spectra and decay of the anisotropy of CK derivatized on a single subunit with the fluorescent sulfhydryl reagent 5-[2-(iodoacetyl)amino-ethyl]aminonaphthalene-1-sulfonate indicated that the derivatized sites are more flexible in the heterodimer. The slow component in the anisotropy decay suggests that hybridization results in a small increase in the packing density or contraction of overall conformation of the B-subunit. The KM for MgATP with singly derivatized CK-MB was the same as the KM for the native enzyme. However, derivatization of a single subunit caused the Vmax to decrease by greater than 50%, which indicates that subunit-subunit interactions may modulate the activity of CK. A model for assembly of CK-MB is proposed which includes subunit characteristics more similar to those found in the muscle-type homodimer than in the brain-type homodimer and increased flexibility of the active site domain of both subunits.

Introduction

The cytoplasmic isozymes of creatine kinase (EC 2.7.3.2) occur in three tissue specific, dimeric forms designated CK-MM (muscle), CK-BB (brain) and CK-MB (heart) [1]. Creatine kinase MM, which is found in heart as well, is both soluble and a particulate constituent of the M-line structure, whereas CK-MB is found only in the soluble part of the myocardial cell [2]. While the M and B subunits of CK are approximately 80% sequence homologous [3], CK-MM and CK-BB have several different physical properties [1, 4], including complete antigenic non-crossreactivity [5]. An interesting question to pose is how the same subunits which constitute the homodimers are able to comply with the structural requirements for formation of the heterodimer. Subunit conformation in the hybrid may be similar to that in the homodimers or adjustments may be required for activity and subunit-subunit stability. Comparisons of the three isozymes then, affords an opportunity to determine the types of conformational adaptations required for subunit hybridization and addresses the more basic issue of the nature of protein-protein interaction.

Several physical characteristics suggest that conformational changes in subunits occur as the result of hybridization, including relative isoelectric points and electrophoretic mobilities [6], fluorescence resonance energy between the two active sites [7, 8, 9] and antigenicities [5]. An important start in understanding subunit interdigitation of CK at the structural level has been made with the determination of the crystallographic three-dimension conformation of the mitochondrial octameric CK [10]. As more structures are detailed, it would be useful to complement these with an analysis of conformational flexibility. In this study, we have compared elements of secondary, tertiary and quaternary structure of CK-MB with the homodimers. Results indicate significant conformational and dynamic changes upon subunit-subunit interaction in the assembly of the heterodimer.

Section snippets

Materials and methods

Rabbit CK-MB from heart [11] and CK-BB from brain [6] were purified as described previously. Rabbit CK-MM was obtained from Boehringer/Mannheim (Indianapolis, IN). Creatine kinase MB was also prepared by in vitro hybridization [7]. Equal activities of CK-MM and CK-BB in 0.05 M Tris-HCl buffer, pH 8.0 and 2 mM DTT were mixed, then treated with sufficient crystalline, enzyme grade urea, to bring the final concentration to 6 M urea. After 1 h at room temperature, buffer was added to the denatured

Purification of CK-MB from rabbit heart and comparison with CK-MB prepared by in vitro hybridization

Creatine kinase MB isolated from rabbit heart and CK-MB prepared by in vitro hybridization exhibited identical electrophoretic mobilities in agarose thin film electrophoresis and on polyacrylamide gels containing SDS. In the latter method, each protein appeared as two closely migrating bands, with molecular weights approximately 40 kDa. Isozymes MM, BB, MB (purified) and MB (prepared by hybridization) exhibited elution times (±0.01 min) of 6.57, 6.54, 6.59, and 6.59 min, respectively, on an

Discussion

In this investigation, we identify specific conformational changes at the secondary and tertiary levels of protein organization in response to subunit hybridization. The spectral characteristics of CK-MB, whether absorbance, fluorescence or circular dichroism, are not an average of those of CK-MM and CK-BB. Had the conformation of the subunits in the hybrid of CK-MB been the same as those in the homodimers, the difference in absorbance between CK-MB and a mixture of CK-MM+CK-BB should be

Acknowledgements

Supported in part by a grant from the American Heart Association, Florida Affiliate.

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