Binding model of human coactosin-like protein with filament actin revealed by mutagenesis.
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.
Dai
Haiming
H
Hefei National Laboratory for Physical Sciences at Microscale, and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, China.
Huang
Wei
W
Xu
Jian
J
Yao
Bo
B
Xiong
Shangmin
S
Ding
Husheng
H
Tang
Yajun
Y
Liu
Haiyan
H
Wu
Jihui
J
Shi
Yunyu
Y
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
D000199:Actins; C118115:COTL1 protein, human; D017931:DNA Primers; D008840:Microfilament Proteins