Electrospray ionization mass spectrometry studies of noncovalent myosin VI complexes reveal a new specific calmodulin binding site.
Among the myosin superfamily, myosin VI differs from all others by a reverse directionality and a particular motility. Little structural information is available for myosin VI. It is known that it binds one calmodulin (CaM) by means of a single "IQ motif" and that myosin VI contains a specific insert located at the junction between the motor domain (MD) and the lever arm, likely to play a critical role for the unusual motility previously observed. Electrospray ionization mass spectrometry (MS) was used to determine the CaM and Ca2+ stoichiometries in several myosin VI constructs. In particular, the experimental conditions required for the observation of multiprotein/Ca2+ noncovalent assemblies are detailed for two truncated MD constructs (less than 20 kDa) and for three full MD constructs (more than 90 KDa). The specificity of the detected stoichiometries is discussed for each construct and the resolving power of Time of Flight mass spectrometry is stressed, in particular for the detection of metal ions binding to high molecular weight complexes. MS reveals a new CaM binding site for myosin VI and highlights a different behavior for the five myosin VI constructs versus Ca2+ binding. In addition to these stoichiometry based experiments, gas-phase dissociation analyses on intact complexes are described. They reveal that Ca2+ transfer between protein partners occurs during the dissociation process for one construct with a full MD. Charge-transfer and dissociation behavior has allowed to draw structural assumptions for the interaction of the MD with the CaM N-terminal lobe.
Laboratoire de Spectrométrie de Masse Bio-Organique, Strasbourg, France.
Guillaume
G
Chevreux
Noelle
N
Potier
Alain
A
Van Dorsselaer
Amel
A
Bahloul
Anne
A
Houdusse
Amber
A
Wells
H Lee
HL
Sweeney
D000818:Animals; D001665:Binding Sites; D002147:Calmodulin / Q000378:metabolism*; D018076:DNA, Complementary; D016296:Mutagenesis; D018995:Myosin Heavy Chains / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021241:Spectrometry, Mass, Electrospray Ionization; D013552:Swine
D002147:Calmodulin; D018076:DNA, Complementary; C089785:myosin VI; D018995:Myosin Heavy Chains