The NMR solution structure of human glutaredoxin in the fully reduced form

Abstract

The determination of the nuclear magnetic resonance (NMR) solution structure of fully reduced human glutaredoxin is described. A total of 1159 useful nuclear Overhauser effect (NOE) upper distance constraints and 187 dihedral angle constraints were obtained as the input for the structure calculations for which the torsion angle dynamics program DYANA has been utilized followed by energy minimization in water with the AMBER force ®eld as implemented in the program OPAL. The resulting 20 conformers have an average root-mean-square deviation value relative to the mean coordinates of 0.54 A Ê for all the backbone atoms N, C a and C H , and of 1.01 A Ê for all heavy atoms. Human glutaredoxin consists of a four-stranded mixed b-sheet composed of residues 15 to 19, 43 to 47, 72 to 75 and 78 to 81, and ®ve a-helices composed of residues 4 to 9, 24 to 34, 54 to 65, 83 to 91, and 94 to 100. Comparisons with the structures of Escherichia coli glutaredoxin-1, pig liver glutaredoxin and human thioredoxin were made. Electrostatic calculations on the human glutaredoxin structure and that of related proteins provide an understanding of the variation of pK a values for the nucleophilic cysteine in the active site observed among these proteins. In addition, the high-resolution NMR solution structure of human glutaredoxin has been used to model the binding site for glutathione and for ribonucleotide reductase B1 by molecular dynamics simulations.

Oxidation and S-Nitrosylation of Cysteines in Human Cytosolic and Mitochondrial Glutaredoxins

“…Structural changes could also inactivate the protein. Cys-8 is located in the N terminus and is solvent-exposed in the structure of reduced Grx1 (31). It contributes significantly to the tendency of Grx1 to precipitate in solution, and the corresponding Cys to Ser mutant improved this behavior (26).…”

“…This protein does not contain a Cys residue corresponding to Cys-8 in human Grx1, and both Cys residues corresponding to Cys-79 and Cys-83 are present in the thiol form. Cys-83 is located close to the active site in an area important for substrate binding and has previously been suggested to be a potential site for regulation (31); Cys-79 is located at the end of ␤-sheet 4. Both are solvent-exposed but separated by a distance of ϳ12 Å (c␣-c␣) in the structures of mammalian Grx1s (29,31).…”

“…Cys-83 is located close to the active site in an area important for substrate binding and has previously been suggested to be a potential site for regulation (31); Cys-79 is located at the end of ␤-sheet 4. Both are solvent-exposed but separated by a distance of ϳ12 Å (c␣-c␣) in the structures of mammalian Grx1s (29,31). Formation of a disulfide bond between these two cysteines would certainly have a profound effect on the structure of the protein.…”

Comparative analysis of glutaredoxin domains from bacterial opportunistic pathogens

“…This might kill bacteria by inhibition of DNA synthesis and/or through increases in ROS toxicity. Structures have been published for several forms of human (Sun et al, 1998;Yang et al, 1998), plant (Rouhier et al, 2007;Li et al, 2010), budding yeast (Gibson et al, 2008;Discola et al, 2009) and Escherichia coli GLXRs (Iwema et al, 2009;Fladvad et al, 2005;Xia et al, 1992Xia et al, , 2001Bushweller et al, 1994;Sodano et al, 1991). However, it was unclear whether other bacterial GLXRs would adopt similar conformations.…”

Human Mitochondrial Glutaredoxin Reduces S-Glutathionylated Proteins with High Affinity Accepting Electrons from Either Glutathione or Thioredoxin Reductase

“…The two Grxs show only 34% sequence identity, and the proteins differ both in size and active site sequence. The 12-kDa cytosolic Grx1 has been extensively studied (18 -29), and three-dimensional structures have been determined for the reduced form and the glutathione-mixed disulfide intermediate (27,30,31). Grx1 is an electron donor for ribonucleotide reductase (5,6,24) and is involved in many different cellular processes like dehydroascorbate reduction (32), actin polymerization (33,34), protection against oxidative stress (35,36), apoptosis after acute cadmium exposure (37), and cellular differentiation (38).…”