Mutation of the pseudo‐EF‐hand of calbindin D<sub>9k</sub> into a normal EF‐hand

Johansson, Charlotta; Brodin, Peter; Grundström, Thomas; Forsén, Sture; Drakenberg, T.

doi:10.1111/j.1432-1033.1991.tb16501.x

Cited by 16 publications

(14 citation statements)

References 28 publications

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“…The F36G mutant was produced using cassette mutagenesis, overexpressed in Escherichia coli , and purified (Linse et al 1987; Johansson et al 1990). This mutant also contained the mutation of Pro 43 to methionine.…”

Section: Methodsmentioning

confidence: 99%

The EF‐hand domain: A globally cooperative structural unit

et al. 2002

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EF-hand Ca2+ -binding proteins participate in both modulation of Ca 2+ signals and direct transduction of the ionic signal into downstream biochemical events. The range of biochemical functions of these proteins is correlated with differences in the way in which they respond to the binding of Ca 2+. The EF-hand domains of calbindin D 9k and calmodulin are homologous, yet they respond to the binding of calcium ions in a drastically different manner. A series of comparative analyses of their structures enabled the development of hypotheses about which residues in these proteins control the calcium-induced changes in conformation. To test our understanding of the relationship between protein sequence and structure, we specifically designed the F36G mutation of the EF-hand protein calbindin D 9k to alter the packing of helices I and II in the apoprotein. The three-dimensional structure of apo F36G was determined in solution by nuclear magnetic resonance spectroscopy and showed that the design was successful. Surprisingly, significant structural perturbations also were found to extend far from the site of mutation. The observation of such long-range effects provides clear evidence that four-helix EF-hand domains should be treated as a single globally cooperative unit. A hypothetical mechanism for how the long-range effects are transmitted is described. Our results support the concept of energetic and structural coupling of the key residues that are crucial for a protein's fold and function.Keywords: Calcium-binding protein; conformational change; EF-hand; mutagenesis; NMR spectroscopy; protein engineeringThe EF-hand family of Ca 2+ -binding proteins (CaBPs) provides a rich framework for investigating fundamental relationships between protein sequence and biochemical function. The EF-hand motif is among the most common in animal cells (Henikoff et al. 1997); more than 1000 have been identified from their unique sequence signatures. These motifs are organized into structural units/domains containing two or more EF hands that form highly stable helical bundles (Nelson and Chazin 1998a). Despite the similarities in their sequences and structures, EF-hand proteins perform a diverse range of functions. There are two primary classes of EF-hand proteins: Ca 2+ sensors, which transduce Ca 2+ signals, and Ca 2+ signal modulators, which modulate the shape and/or duration of Ca 2+ signals or participate in Ca 2+ homeostasis. Here, we describe research on representative sensor and signal modulator proteins, calmodulin (CaM) and calbindin D 9k (calbindin), which is directed toward understanding how differences in amino acid sequence specify differences in how EF-hand domains re-

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Section: Methodsmentioning

confidence: 99%

The EF‐hand domain: A globally cooperative structural unit

et al. 2002

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“…Changing loop I into a normal EF-hand loop, either by inserting the sequence of loop II or by the minimum number of replacements and deletions, did not result in a 113 Cd spectrum expected for two typical EF-hands, but a shift for site I 113 Cd of 20–30 ppm downfield from the expected value (Figure 6). This shows that the general structure around loop I does not easily adopt a typical EF-hand loop [34,37]. …”

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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“…This last mutant has a drastic charge change (from 11 to ÿ3 in the presence of calcium) that should invoke significant net repulsion of the negatively charged partner subdomain EF2. Intact proteins with the A15D1P20G, and E17Q1D19N substitutions have been studied before (Akke and Forsén;Johansson et al, 1991;Linse et al, 1988Linse et al, , 1991, whereas the other three mutants were designed specifically for this study. The extra P20G substitution provides A15D1P20G with a Ca 21 affinity that is only twofold reduced compared to wild-type (Johansson et al, 1991).…”

Section: Mutagenesis and Nomenclaturementioning

confidence: 99%

Electrostatic Contributions to the Kinetics and Thermodynamics of Protein Assembly

Dell’Orco¹,

Xue

Thulin

et al. 2005

Biophysical Journal

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The role of electrostatic interactions in the assembly of a native protein structure was studied using fragment complementation. Contributions of salt, pH, or surface charges to the kinetics and equilibrium of calbindin D(9k) reconstitution was measured in the presence of Ca(2+) using surface plasmon resonance and isothermal titration calorimetry. Whereas surface charge substitutions primarily affect the dissociation rate constant, the association rates are correlated with subdomain net charge in a way expected for Coulomb interactions. The affinity is reduced in all mutants, with the largest effect (260-fold) observed for the double mutant K25E+K29E. At low net charge, detailed charge distribution is important, and charges remote from the partner EF-hand have less influence than close ones. The effects of salt and pH on the reconstitution are smaller than mutational effects. The interaction between the wild-type EF-hands occurs with high affinity (K(A) = 1.3 x 10(10) M(-1); K(D) = 80 pM). The enthalpy of association is overall favorable and there appears to be a very large favorable entropic contribution from the desolvation of hydrophobic surfaces that become buried in the complex. Electrostatic interactions contribute significantly to the affinity between the subdomains, but other factors, such as hydrophobic interactions, dominate.

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Mutation of the pseudo‐EF‐hand of calbindin D_9k into a normal EF‐hand

Cited by 16 publications

References 28 publications

The EF‐hand domain: A globally cooperative structural unit

The EF‐hand domain: A globally cooperative structural unit

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

Electrostatic Contributions to the Kinetics and Thermodynamics of Protein Assembly

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Mutation of the pseudo‐EF‐hand of calbindin D9k into a normal EF‐hand

Cited by 16 publications

References 28 publications

The EF‐hand domain: A globally cooperative structural unit

The EF‐hand domain: A globally cooperative structural unit

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

Electrostatic Contributions to the Kinetics and Thermodynamics of Protein Assembly

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Mutation of the pseudo‐EF‐hand of calbindin D_9k into a normal EF‐hand