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Nef of HIV-1 interacts directly with calcium-bound calmodulin

¹ Division of Biomedical Polymer Science, Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi 470-1192, Japan
² Graduate School of Engineering, Yamagata University, Yonezawa 992-8510, Japan
³ School of Health Sciences, Sapporo Medical University, Sapporo 060-8556, Japan

Reprint requests to: Nobuhiro Hayashi, Ph.D., Division of Biomedical Polymer Science, Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi 470-1192, Japan; e-mail: nhayashi{at}fujita-hu.ac.jp; fax: 81-562-93-8832.

It was recently found that the myristoyl group of CAP-23/NAP-22, a neuron-specific protein kinase C substrate, is essential for the interaction between the protein and Ca²⁺-bound calmodulin (Ca²⁺/CaM). Based on the N-terminal amino acid sequence alignment of CAP-23/NAP-22 and other myristoylated proteins, including the Nef protein from human immunodeficiency virus (HIV), we proposed a new hypothesis that the protein myristoylation plays important roles in protein–calmodulin interactions. To investigate the possibility of direct interaction between Nef and calmodulin, we performed structural studies of Ca²⁺/CaM in the presence of a myristoylated peptide corresponding to the N-terminal region of Nef. The dissociation constant between Ca²⁺/CaM and the myristoylated Nef peptide was determined to be 13.7 nM by fluorescence spectroscopy analyses. The NMR experiments indicated that the chemical shifts of some residues on and around the hydrophobic clefts of Ca²⁺/CaM changed markedly in the Ca²⁺/CaM-Nef peptide complex with the molar ratio of 1:2. Correspondingly, the radius of gyration determined by the small angle X-ray scattering measurements is 2–3 Å smaller that of Ca²⁺/CaM alone. These results demonstrate clearly that Nef interacts directly with Ca²⁺/CaM.

Keywords: Myristoylation; calmodulin; HIV-1 Nef; NMR; protein protein interaction

Abbreviations: HIV, human immunodeficiency virus • CaM, calmodulin • Ca²⁺/CaM, Ca²⁺-bound calmodulin • Nef peptide, myristoylated peptides corresponding to the N-terminal region of HIV-1 Nef • SAXS, small angle X-ray scattering • NMR, nuclear magnetic resonance spectroscopy • mC/N9, myristoylated peptide corresponding to the N-terminal CaM binding site of CAP-23/NAP-22 • N, -amino • MAP kinase, mitogen-activated protein kinase • HSQC, heteronuclear single quantum coherence • MLCK, myosin light chain kinase • M13, a peptide based on the calmodulin-binding domain of MLCK • PGD, petunia glutamate decarboxylase

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