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

Abstract

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.

Metal Exchange in the Interprotein ZnII‐Binding Site of the Rad50 Hook Domain: Structural Insights into CdII‐Induced DNA‐Repair Inhibition

“…[11,12] Structure-interfering properties of Cd II bindingt oZ n IIcontaining proteins remain controversial because ford ecades Cd II was used as aZ n II -isostructural spectroscopic probe for Zn II -binding proteins in UV spectrophotometric studies and 113 Cd NMR spectroscopy. [13][14][15][16][17] Although Cd II and Zn II ionicr adii are relatively similar and displacementi su sually one-to-one, in terms of stoichiometry and coordination sphere, their binding to at arget protein could potentially generate ad ifferent structural outcome. The presented scientific problem of Zn II -to-Cd II substitution and its impact on zinc-binding proteins have been extensively studied;h owever,a ll published data are focused on intramolecular sites, mostly of zinc finger domains and metallothioneins.…”

Chronic inflammation role in the obesity-diabetes association: a case-cohort study

“…The World Health Organization estimates that 2.9 million deaths per year are attributable to diabetes, and this number is likely to increase by more than 50% in the next decade [1]. Much of this burden may be explained by the dramatic rise in obesity rates [2].…”

Cd2+as a Ca2+Surrogate in Protein–Membrane Interactions: Isostructural but Not Isofunctional

“…15 In this work, we investigated the effect of Cd 2+ on the structure and membrane-binding properties of the C2 domain from protein kinase Cα (C2α). Unexpectedly, we found that despite high affinity of Cd 2+ to C2α and the similar coordination geometry of protein-bound Cd 2+ and Ca 2+ , Cd 2+ is unable to support the membrane-binding function of the protein.…”