The denuded IQ2 domain, i.e. myosin heavy chain not associated with regulatory light chains, exerts an inhibitory effect on myosin ATPase activity. In this study, we elaborated a structural explanation for this auto-inhibitory effect of IQ2 on myosin function. We employed analytical ultracentrifugation, circular dichroism, and surface plasmon resonance spectroscopy to investigate structural and functional properties of a myosin heavy chain (MYH) head-rod fragment aa664-915. MYH664-915 was monomeric, adopted a closed shape, and bound essential myosin light chains (HIS-MLC-1) with low affinity to IQ1. Deletion of IQ2, however opened MYH664-915. Four amino acids present in IQ2 could be identified to be responsible for this auto-inhibitory structural effect: alanine mutagenesis of 1814, Q815, R819, and W827 stretched MYH664-915 and increased 30-fold the binding affinity of HIS-MLC-1 to IQ1. In this study we show, that denuded IQ2 favours a closed conformation of myosin with a low HIS-MLC-1 binding affinity. The collapsed structure of myosin with denuded IQ2 could explain the auto-inhibitory effects of IQ2 on enzymatic activity of myosin. (C) 2010 Elsevier Inc. All rights reserved.