Domain stability and metal-induced folding of calcium- and integrin-binding protein 1.
It is widely accepted that a pair of EF-hands is the functional unit of typical four EF-hand proteins such as calmodulin or troponin C. In this work we investigate the structure and stability of the four EF-hand domains in the related protein calcium- and integrin-binding protein 1 (CIB1) in the presence and absence of Mg2+ or Ca2+, to determine if similar EF-hand interactions occur. The backbone structure and flexibility of CIB1 were first studied by NMR spectroscopy, and these studies were complimented with steady-state fluorescence spectroscopy and chemical denaturation experiments using mutant CIB1 proteins having single Trp reporter groups in each of the four EF-hand domains EF-I (F34W), EF-II (F91W), EF-III (L128W), and EF-IV (F173W). We find that Mg2+-CIB1 adopts a well-folded structure similar to Ca2+-CIB1, except for some conformational heterogeneity in the C-terminal EF-IV domain. The structure of apo-CIB1 is significantly more dynamic, especially within EF-II, EF-III, and a partially unfolded EF-IV region, but the N-terminal EF-I region of apo-CIB1 has a well-ordered and more stable structure. The data reveal significant communication between the N- and C-lobes of CIB1, and show that transient intermediate conformations are formed along the unfolding pathway for each form of the protein. Collectively the data demonstrate that the communication between the paired EF-hand domains as well as between the N- and C-lobes of CIB1 is distinct from the ancestral proteins calmodulin and troponin C, which might be important for the unique function of CIB1 in numerous biological processes.
Yamniuk
Aaron P
AP
Structural Biology Research Group, Department of Biological Sciences, University of Calgary, 2500 University Drive N.W., Calgary, AB, Canada, T2N 1N4.
Silver
Dylan M
DM
Anderson
Kathryn L
KL
Martin
Stephen R
SR
Vogel
Hans J
HJ
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004355:Drug Stability; D006801:Humans; D066298:In Vitro Techniques; D008274:Magnesium / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry
C109198:CIB1 protein, human; D002135:Calcium-Binding Proteins; D011994:Recombinant Proteins; D014364:Tryptophan; D008274:Magnesium; D002118:Calcium