Metal-dependent stabilization of an active HMG protein.
Using a cloned single domain of the high mobility group protein 1 (HMGB1), we evaluated the effect of introducing metal binding site(s) on protein stability and function. An HMG domain is a conserved sequence of approximately 80 amino acids rich in basic, aromatic and proline residues that is active in binding DNA in a sequence- or structure-specific manner. The design strategy focuses on anchoring selected regions of the protein, specifically loops and turns in the molecule, using His-metal ligands. Changes in secondary structure, thermostability and DNA binding properties of a series of such mutants were evaluated. The two most stable mutant constructs contain three surface histidine replacements (two metal binding sites) in the regions encompassing both turns of the molecule. On ligation with the divalent nickel cation, the stability of these two triple histidine mutants (I38H/N51H/D55H and G39H/N51H/D55H) increases by 1.3 and 1.6 kcal/mol, respectively, relative to the wild-type protein, although the creation of binding sites per se destabilizes the protein. The DNA-binding properties of the modified proteins are not impaired by the introduction of the metal binding motifs. These results indicate that it is feasible to stabilize protein tertiary structure using appropriate placement of surface His-metal bonds without loss of function.
Bell
Anthony J
AJ
Department of Chemistry, New York University, New York, NY 10003, USA.
Xin
Hong
H
Taudte
Susann
S
Shi
Zhengshuang
Z
Kallenbach
Neville R
NR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D017124:Conserved Sequence; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D024243:HMGB1 Protein / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D006639:Histidine / Q000737:chemistry / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009532:Nickel / Q000737:chemistry*; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
D004268:DNA-Binding Proteins; D024243:HMGB1 Protein; D011994:Recombinant Proteins; D006639:Histidine; D009532:Nickel; D004247:DNA