Structure, dynamics and thermodynamics of the human centrin 2/hSfi1 complex.
Centrin, an EF-hand calcium-binding protein, has been shown to be involved in the duplication of centrosomes, and Sfi1 (Suppressor of fermentation-induced loss of stress resistance protein 1) is one of its centrosomal targets. There are three isoforms of human centrin, but here we only considered centrin 2 (HsCen2). This protein has the ability to bind to any of the approximately 25 repeats of human Sfi1 (hSfi1) with more or less affinity. In this study, we mainly focused on the 17th repeat (R17-hSfi1-20), which presents the highest level of similarity with a well-studied 17-residue peptide (P17-XPC) from human xeroderma pigmentosum complementation group C protein, another centrin target for DNA repair. The only known structure of HsCen2 was resolved in complex with P17-XPC. The 20-residue peptide R17-hSfi1-20 exhibits the motif L8L4W1, which is the reverse of the XPC motif, W1L4L8. Consequently, the dipole of the helix formed by this motif has a reverse orientation. We wished to ascertain the impact of this reversal on the structure, dynamics and affinity of centrin. To address this question, we determined the structure of C-HsCen2 [the C-terminal domain of HsCen2 (T94-Y172)] in complex with R17-hSfi1-20 and monitored its dynamics by NMR, after having verified that the N-terminal domain of HsCen2 does not interact with the peptide. The structure shows that the binding mode is similar to that of P17-XPC. However, we observed a 2 -A translation of the R17-hSfi1-20 helix along its axis, inducing less anchorage in the protein and the disruption of a hydrogen bond between a tryptophan residue in the peptide and a well-conserved nearby glutamate in C-HsCen2. NMR dynamic studies of the complex strongly suggested the existence of an unusual calcium secondary binding mode in calcium-binding loop III, made possible by the uncommon residue composition of this loop. The secondary metal site is only populated at high calcium concentration and depends on the type of bound ligand.
Martinez-Sanz
Juan
J
Institut Curie-Centre de Recherche, F-91405 Orsay Cedex, France.
Kateb
Fatiha
F
Assairi
Liliane
L
Blouquit
Yves
Y
Bodenhausen
Geoffrey
G
Abergel
Daniel
D
Mouawad
Liliane
L
Craescu
Constantin T
CT
D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D002151:Calorimetry; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000378:metabolism*; D018360:Crystallography, X-Ray; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D017433:Protein Structure, Secondary; D020449:Repetitive Sequences, Amino Acid; D012996:Solutions; D013816:Thermodynamics
C472752:CETN2 protein, human; D002135:Calcium-Binding Proteins; D018797:Cell Cycle Proteins; D010455:Peptides; C545864:Sfi1 protein, human; D012996:Solutions; D002118:Calcium