1QX2

X-ray Structure of Calcium-loaded Calbindomodulin (A Calbindin D9k Re-engineered to Undergo a Conformational Opening) at 1.44 A Resolution

Summary for 1QX2

• Entry DOI: 10.2210/pdb1qx2/pdb
• Descriptor: Vitamin D-dependent calcium-binding protein, intestinal, CALCIUM ION, ZINC ION, ... (4 entities in total)
• Functional Keywords: ef-hand (helix-loop-helix) calcium binding protein, four-helix domain, protein engineering, calcium-induced conformational response, calmodulin, calbindin d9k, signaling protein
• Biological source: Bos taurus (cattle)
• Total number of polymer chains: 2
• Total formula weight: 17328.68

Authors

Bunick, C.G.,Nelson, M.R.,Mangahas, S.,Mizoue, L.S.,Bunick, G.J.,Chazin, W.J. (deposition date: 2003-09-04, release date: 2004-05-25, Last modification date: 2021-10-27)

Primary citation

Bunick, C.G.,Nelson, M.R.,Mangahas, S.,Hunter, M.J.,Sheehan, J.H.,Mizoue, L.S.,Bunick, G.J.,Chazin, W.J.
Designing Sequence to Control Protein Function in an EF-Hand Protein
J.Am.Chem.Soc., 126:5990-5998, 2004

PubMed Abstract: The extent of conformational change that calcium binding induces in EF-hand proteins is a key biochemical property specifying Ca(2+) sensor versus signal modulator function. To understand how differences in amino acid sequence lead to differences in the response to Ca(2+) binding, comparative analyses of sequence and structures, combined with model building, were used to develop hypotheses about which amino acid residues control Ca(2+)-induced conformational changes. These results were used to generate a first design of calbindomodulin (CBM-1), a calbindin D(9k) re-engineered with 15 mutations to respond to Ca(2+) binding with a conformational change similar to that of calmodulin. The gene for CBM-1 was synthesized, and the protein was expressed and purified. Remarkably, this protein did not exhibit any non-native-like molten globule properties despite the large number of mutations and the nonconservative nature of some of them. Ca(2+)-induced changes in CD intensity and in the binding of the hydrophobic probe, ANS, implied that CBM-1 does undergo Ca(2+) sensorlike conformational changes. The X-ray crystal structure of Ca(2+)-CBM-1 determined at 1.44 A resolution reveals the anticipated increase in hydrophobic surface area relative to the wild-type protein. A nascent calmodulin-like hydrophobic docking surface was also found, though it is occluded by the inter-EF-hand loop. The results from this first calbindomodulin design are discussed in terms of progress toward understanding the relationships between amino acid sequence, protein structure, and protein function for EF-hand CaBPs, as well as the additional mutations for the next CBM design.

PubMed: 15137763
DOI: 10.1021/ja0397456

Experimental method

X-RAY DIFFRACTION (1.44 Å)