title: Binding model of human coactosin-like protein with filament actin revealed by mutagenesis.
abstract: Human coactosin-like protein (CLP) is a small (MW approximately 17 kDa) evolutionarily conserved actin-binding protein. It can bind to actin filaments but not globular actin and belongs to the fourth class of ADF-H-domain-containing proteins. Human CLP can also bind to 5LO, which plays an important role in cellular leukotriene synthesis. Although the structure of hCLP has been determined by both NMR and X-ray experiments, how hCLP binds to the actin filament is still a controversial question. To obtain insights into the structure of the complex, we studied the three-dimensional structure and backbone dynamics of hCLP using multidimensional NMR spectroscopy. Guided by the solution structure of the protein, a series of site-directed mutants were generated and their F-actin-binding activities were measured by high-speed cosedimentation assays. Furthermore, the structure model of the hCLP-F-actin complex was proposed using computational docking with the docking results filtered by the mutation data. Several previously untested residues (including T66, L89, R91, K102, D116 and E119) in hCLP were found important for the F-actin-binding activity. The extended region of beta4-beta5 of hCLP (residue 66-75) was found very flexible and very important for F-actin binding. The C-terminal residues of hCLP were not involved in F-actin binding, which was different from UNC-60B. Based on our hCLP-F-actin-binding model, different affinities of the four classes of ADF-H domain containing proteins for F-actin were explained.
authors.affiliation: Hefei National Laboratory for Physical Sciences at Microscale, and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, China.
authors.forename: Haiming
authors.initials: H
authors.lastname: Dai
authors.forename: Wei
authors.initials: W
authors.lastname: Huang
authors.forename: Jian
authors.initials: J
authors.lastname: Xu
authors.forename: Bo
authors.initials: B
authors.lastname: Yao
authors.forename: Shangmin
authors.initials: S
authors.lastname: Xiong
authors.forename: Husheng
authors.initials: H
authors.lastname: Ding
authors.forename: Yajun
authors.initials: Y
authors.lastname: Tang
authors.forename: Haiyan
authors.initials: H
authors.lastname: Liu
authors.forename: Jihui
authors.initials: J
authors.lastname: Wu
authors.forename: Yunyu
authors.initials: Y
authors.lastname: Shi
mesh_terms: D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002942:Circular Dichroism; D017931:DNA Primers; D006801:Humans; D008840:Microfilament Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013056:Spectrophotometry, Ultraviolet
chemical_list: D000199:Actins; C118115:COTL1 protein, human; D017931:DNA Primers; D008840:Microfilament Proteins