A Calbindin D9k Mutant with Reduced Calcium Affinity and Enhanced Cooperativity. Metal Ion Binding, Stability, and Structural Studies

Linse, Sara; Bylsma, Niels; Drakenberg, Torbjörn; Sellers, Peter; Forsén, Sture; Thulin, Eva; Svensson, L.A.; Zajtzeva, I.; Zajtsev, V.M.; Marek, Jaromı́r

doi:10.1021/bi00207a015

Cited by 11 publications

(21 citation statements)

References 30 publications

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“…The residues of binding site II appeared to be more reactive both in accumulated contact time (up to ∼29% for D58 and ∼40% for E60/D60) and in the number of primary and secondary encounters. These findings are correlated with experimental indications that site II is occupied first during the binding process [22]. Finally, there are residues that are not associated directly with the specific binding sites (P43, D47, E48, E51) that are attractive both for primary and secondary encounters, suggesting that they may function in passing the ion between nearby residues.…”

Section: Resultssupporting

confidence: 73%

“…This conservative mutation has a highly similar structure to the WT CaB, yet at physiologic ionic strength its affinity to Ca 2+ is reduced by ∼8 fold [22]. This effect was partly attributed [22] to a more labile water molecule that binds site I Ca 2+ (the left binding site, as seen in Figures 6). As shown in Figure 6B, the side chain of E60, as determined for the crystalline protein, indirectly coordinates the Ca 2+ ion in site I via a water molecule.…”

Section: Resultsmentioning

confidence: 92%

“…What is more, the experimental measurements on CaB mutants can be used as references for simulations on similar mutants. In the present study, we performed 100 ns long simulation of the WT protein and the E60D mutant, which is known experimentally to have a reduced affinity to Ca 2+ [22]. The simulations show that in most of the time the Ca 2+ ion spends within the Debye radius of CaB, it is detained at the 2 nd and 1 st solvation shells.…”

Section: Introductionmentioning

confidence: 93%

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The Dynamics of Ca2+ Ions within the Solvation Shell of Calbindin D9k

2011

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The encounter of a Ca2+ ion with a protein and its subsequent binding to specific binding sites is an intricate process that cannot be fully elucidated from experimental observations. We have applied Molecular Dynamics to study this process with atomistic details, using Calbindin D9k (CaB) as a model protein. The simulations show that in most of the time the Ca2+ ion spends within the Debye radius of CaB, it is being detained at the 1st and 2nd solvation shells. While being detained near the protein, the diffusion coefficient of the ion is significantly reduced. However, due to the relatively long period of detainment, the ion can scan an appreciable surface of the protein. The enhanced propagation of the ion on the surface has a functional role: significantly increasing the ability of the ion to scan the protein's surface before being dispersed to the bulk. The contribution of this mechanism to Ca2+ binding becomes significant at low ion concentrations, where the intervals between successive encounters with the protein are getting longer. The efficiency of the surface diffusion is affected by the distribution of charges on the protein's surface. Comparison of the Ca2+ binding dynamics in CaB and its E60D mutant reveals that in the wild type (WT) protein the carboxylate of E60 function as a preferred landing-site for the Ca2+ arriving from the bulk, followed by delivering it to the final binding site. Replacement of the glutamate by aspartate significantly reduced the ability to transfer Ca2+ ions from D60 to the final binding site, explaining the observed decrement in the affinity of the mutated protein to Ca2+.

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Section: Resultssupporting

confidence: 73%

Section: Resultsmentioning

confidence: 92%

Section: Introductionmentioning

confidence: 93%

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The Dynamics of Ca2+ Ions within the Solvation Shell of Calbindin D9k

2011

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“…This is also seen as a decreased cooperativity in the calcium binding. In fact there is a correlation, though weak, between the cooperativity in calcium binding and shift in site II 113 Cd caused by binding of Cd to site I [38,40,41]. …”

Section: Specific Highlights Of Studies On Alkaline Phosphatase Camentioning

confidence: 99%

Use of 113Cd NMR to Probe the Native Metal Binding Sites in Metalloproteins: An Overview

Armitage

Drakenberg

Reilly

2012

Metal Ions in Life Sciences

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Our laboratories have actively published in this area for several years and the objective of this chapter is to present as comprehensive an overview as possible. Following a brief review of the basic principles associated with 113Cd NMR methods, we will present the results from a thorough literature search for 113Cd chemical shifts from metalloproteins. The updated 113Cd chemical shift figure in this chapter will further illustrate the excellent correlation of the 113Cd chemical shift with the nature of the coordinating ligands (N, O, S) and coordination number/geometry, reaffirming how this method can be used not only to identify the nature of the protein ligands in uncharacterized cases but also the dynamics at the metal binding site. Specific examples will be drawn from studies on alkaline phosphatase, Ca2+ binding proteins, and metallothioneins. In the case of Escherichia coli alkaline phosphatase, a dimeric zinc metalloenzyme where a total of six metal ions (three per monomer) are involved directly or indirectly in providing the enzyme with maximal catalytic activity and structural stability, 113Cd NMR, in conjunction with 13C and 31P NMR methods, were instrumental in separating out the function of each class of metal binding sites. Perhaps most importantly, these studies revealed the chemical basis for negative cooperativity that had been reported for this enzyme under metal deficient conditions. Also noteworthy was the fact that these NMR studies preceeded the availability of the X-ray crystal structure. In the case of the calcium binding proteins, we will focus on two proteins: calbindin D9k and calmodulin. For calbindin D9k and its mutants, 113Cd NMR has been useful both to follow actual changes in the metal binding sites and the cooperativity in the metal binding. Ligand binding to calmodulin has been studied extensively with 113Cd NMR showing that the metal binding sites are not directly involved in the ligand binding. The 113Cd chemical shifts are, however, exquisitely sensitive to minute changes in the metal ion environment. In the case of metallothionein, we will reflect upon how 113Cd substitution and the establishment of specific Cd to Cys residue connectivity by proton-detected heteronuclear 1H-113Cd multiple-quantum coherence methods (HMQC) was essential for the initial establishment of the 3D structure of metallothioneins, a protein family deficient in the regular secondary structural elements of α-helix and β-sheet and the first native protein identified with bound Cd. The 113Cd NMR studies also enabled the characterization of the affinity of the individual sites for 113Cd and, in competition experiments, for other divalent metal ions: Zn, Cu, and Hg.

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“…Cd 2+ , for which MOPAC semi‐empirical parameters are known, was used as the metal ion (the parameters for the Ca 2+ ion are not available) [16]. Cd 2+ has been shown to be a good substitute for Ca 2+ [17–22]. The side‐chains for residues 7, 7′, 8, 8′, 9 and 9′, the NH moiety for residues 7 and 7′, the C=O moiety for residues 9 and 9′ and the C(γ) atom for the side‐chain carboxylate groups in position 12 and 12′ were substituted by hydrogen atoms in the model cycle.…”

Section: Methodsmentioning

confidence: 99%

Cooperative cyclic interactions involved in metal binding to pairs of sites in EF‐hand proteins

Biekofsky¹,

Feeney²

1998

FEBS Letters

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This study focuses on a closed net of electron-pair donor^acceptor interactions, present in the core of all metalbound EF-hand pairs, that link both metal ions across a short two-stranded L L-sheet. A molecular model based on the above cycle of interactions was studied using semi-empirical molecular orbital quantum mechanical methods. The calculations indicate that the interactions in the model cycle are cooperative, that is, that the interaction energy of the cyclic structure is greater than that of the sum of isolated interactions between its components. The cooperativity in this cycle can be attributed to an increase in the stability of the interactions resulting from a mutual polarisation of the associated groups. The predicted polarisation of the amide groups in the cycle is in agreement with experimental NMR IS N deshielding observed for these amide groups upon metal binding. Experimental observations of strengthening of the L L-sheet hydrogen bonds are also consistent with the model calculations. By this mechanism, the binding of the first metal ion would enhance the binding of the second metal ion, and thus, the intradomain cooperativity in cation binding of calmodulin and related EF-hand proteins can be ascribed, at least partly, to this short-range molecular mechanism.z 1998 Federation of European Biochemical Societies.

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A Calbindin D9k Mutant with Reduced Calcium Affinity and Enhanced Cooperativity. Metal Ion Binding, Stability, and Structural Studies

Cited by 11 publications

References 30 publications

The Dynamics of Ca2+ Ions within the Solvation Shell of Calbindin D9k

The Dynamics of Ca2+ Ions within the Solvation Shell of Calbindin D9k

Use of 113Cd NMR to Probe the Native Metal Binding Sites in Metalloproteins: An Overview

Cooperative cyclic interactions involved in metal binding to pairs of sites in EF‐hand proteins

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