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¹¹³Cd?NMR experiments reveal an unusual metal cluster in the solution structure of the yeast splicing protein Bud31p.

ABSTRACT: Establishing the binding topology of structural zinc ions in proteins is an essential part of their structure determination by NMR spectroscopy. Using (113)Cd?NMR experiments with (113)Cd-substituted samples is a useful approach but has previously been limited mainly to very small protein domains. Here we used (113)Cd?NMR spectroscopy during structure determination of Bud31p, a 157-residue yeast protein containing an unusual Zn3Cys9 cluster, demonstrating that recent hardware developments make this approach feasible for significantly larger systems.

SUBMITTER: van Roon AM

PROVIDER: S-EPMC4471582 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature

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¹¹³Cd NMR experiments reveal an unusual metal cluster in the solution structure of the yeast splicing protein Bud31p.

van Roon Anne-Marie M AM Yang Ji-Chun JC Mathieu Daniel D Bermel Wolfgang W Nagai Kiyoshi K Neuhaus David D

Angewandte Chemie (International ed. in English) 20150220 16

Establishing the binding topology of structural zinc ions in proteins is an essential part of their structure determination by NMR spectroscopy. Using (113)Cd NMR experiments with (113)Cd-substituted samples is a useful approach but has previously been limited mainly to very small protein domains. Here we used (113)Cd NMR spectroscopy during structure determination of Bud31p, a 157-residue yeast protein containing an unusual Zn3Cys9 cluster, demonstrating that recent hardware developments make t ...[more]

PMID: 25703931

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Project description: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 (113)Cd NMR methods, we will present the results from a thorough literature search for (113)Cd chemical shifts from metalloproteins. The updated (113)Cd chemical shift figure in this chapter will further illustrate the excellent correlation of the (113)Cd 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, Ca(2+) 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, (113)Cd NMR, in conjunction with (13)C and (31)P 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 preceded the availability of the X-ray crystal structure.In the case of the calcium binding proteins, we will focus on two proteins: calbindin D(9k) and calmodulin. For calbindin D(9k) and its mutants, (113)Cd 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 (113)Cd NMR showing that the metal binding sites are not directly involved in the ligand binding. The (113)Cd chemical shifts are, however, exquisitely sensitive to minute changes in the metal ion environment.In the case of metallothionein, we will reflect upon how (113)Cd substitution and the establishment of specific Cd to Cys residue connectivity by proton-detected heteronuclear (1)H-(113)Cd 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 (113)Cd NMR studies also enabled the characterization of the affinity of the individual sites for (113)Cd and, in competition experiments, for other divalent metal ions: Zn, Cu, and Hg.

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¹¹³Cd?NMR experiments reveal an unusual metal cluster in the solution structure of the yeast splicing protein Bud31p.

Publications

¹¹³Cd NMR experiments reveal an unusual metal cluster in the solution structure of the yeast splicing protein Bud31p.

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