Characterization of the structure and self-recognition of the human centrosomal protein NA14: implications for stability and function.
The protein NA14 is a key adaptor protein mediating the intermolecular interactions of microtubules and Spastin. To gain insight into its structure and function, we have expressed, purified and characterized human NA14 and some variants. NA14 is rather insoluble and tends to oligomerize and form fibrils. Successive mutation of the three Cys and two potentially exposed Leu residues (83 and 93) yielded a water-soluble quintuple variant, named 3CS-2LR. NA14 and its variants have a high helical content as determined by circular dichroism (CD). Based on nuclear magnetic resonance data of the quintuple mutant and the wild-type (wt) protein in the presence of dodecylphosphocholine micelles, the N-(M1-N13) and C-termini (K105-S119) were found to lack preferred structure. The remaining residues (14-104) participate in NA14 self-association, probably by forming a parallel coiled-coil structure. We hypothesize that Leu 83 and Leu 93 mediate interactions among NA14, Spastin and microtubules. We have also examined urea and thermal denaturation of the quintuple and other NA14 variants at different pH values by CD. The pH dependence of the conformational stability and the elevated native-state pK(a) determined for the two conserved Tyr allow us to propose that the NA14 structure may be stabilized by two Glu-COO(-) ||| HO-Tyr H-bonds, highly conserved in NA14-like proteins in other species.
Departamento de Química Física Biológica,  Instituto de Química Física Rocasolano,  CSIC, Serrano-119, 28006 Madrid,  Spain.
Mar
M
Rodríguez-Rodríguez
Miguel A
MA
Treviño
Douglas V
DV
Laurents
Rocío
R
Arranz
José M
JM
Valpuesta
Manuel
M
Rico
Marta
M
Bruix
M Angeles
MA
Jiménez
D000595:Amino Acid Sequence; D000818:Animals; D001324:Autoantigens / Q000737:chemistry* / Q000235:genetics; D018385:Centrosome / Q000378:metabolism; D002942:Circular Dichroism; D006801:Humans; D006860:Hydrogen Bonding; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D016415:Sequence Alignment; D012995:Solubility; D013056:Spectrophotometry, Ultraviolet; D014867:Water
D001324:Autoantigens; C110204:NA14 nuclear autoantigen; D009687:Nuclear Proteins; D014867:Water