Deciphering the zinc coordination properties of the prokaryotic zinc finger domain: The solution structure characterization of Ros87 H42A functional mutant.
The zinc coordination sphere in prokaryotic zinc finger domain is extremely versatile and influences the stability and the folding property of the domain. Of a particular interest is the fourth zinc coordinating position, which is frequently occupied by two successive histidines, both able to coordinate the metal ion. To clarify their structural and functional role we report the NMR solution structure and the dynamics behavior of Ros87 H42A, which is a functional mutant of Ros87, the DNA binding domain of the Ros protein containing a prokaryotic Cys2His2 zinc finger domain. The structural analysis indicates that reducing the spacer among the two coordinating histidines from 4 (among His37 and His42) amino acids to 3 (among His37 and His41) increases the helicity of the first α-helix. At the same time, the second helix appears more mobile in the μs-ms timescale and the hydrophobic core is reduced. These data explain the high frequency of three-residue His spacers in the eukaryotic zinc finger domain and their absence in the prokaryotic counterpart. Furthermore, the structural comparison shows that the second coordination position is more sensitive to H42A mutation with respect to the first and the third position, providing the rationale of the high variability of the second and the fourth zinc coordinating position in Ros homologs, which adopt different metal coordination but preserve similar tertiary structures and DNA binding activities. Finally, H/D exchange measurements and NMR thermal unfolding analysis indicate that this mutant likely unfolds via a different mechanism with respect to the wild-type.
Department of Environmental, Biological and Pharmaceutical Sciences and Technology, Via Vivaldi 43, 81100 Caserta, Italy.
Maddalena
M
Palmieri
Department of Environmental, Biological and Pharmaceutical Sciences and Technology, Via Vivaldi 43, 81100 Caserta, Italy.
Luigi
L
Russo
Department of Environmental, Biological and Pharmaceutical Sciences and Technology, Via Vivaldi 43, 81100 Caserta, Italy.
Gaetano
G
Malgieri
Department of Environmental, Biological and Pharmaceutical Sciences and Technology, Via Vivaldi 43, 81100 Caserta, Italy.
Sabrina
S
Esposito
Department of Environmental, Biological and Pharmaceutical Sciences and Technology, Via Vivaldi 43, 81100 Caserta, Italy.
Ilaria
I
Baglivo
Department of Environmental, Biological and Pharmaceutical Sciences and Technology, Via Vivaldi 43, 81100 Caserta, Italy.
Alessia
A
Rivellino
Interuniversity Centre for Research on Bioactive Peptides (CIRPEB), University of Naples Federico II, Via Mezzocannone 16, 80134 Naples, Italy.
Biancamaria
B
Farina
Institute of Biostructures and Bioimaging, CNR, Via Mezzocannone 16, 80134 Naples, Italy.
Ivan
I
de Paola
Institute of Biostructures and Bioimaging, CNR, Via Mezzocannone 16, 80134 Naples, Italy; Interuniversity Centre for Research on Bioactive Peptides (CIRPEB), University of Naples Federico II, Via Mezzocannone 16, 80134 Naples, Italy.
Laura
L
Zaccaro
Institute of Biostructures and Bioimaging, CNR, Viale A. Doria 6, 95125 Catania, Italy.
Danilo
D
Milardi
Department of Environmental, Biological and Pharmaceutical Sciences and Technology, Via Vivaldi 43, 81100 Caserta, Italy; Interuniversity Centre for Research on Bioactive Peptides (CIRPEB), University of Naples Federico II, Via Mezzocannone 16, 80134 Naples, Italy.
Carla
C
Isernia
Department of Environmental, Biological and Pharmaceutical Sciences and Technology, Via Vivaldi 43, 81100 Caserta, Italy.
Paolo Vincenzo
PV
Pedone
Department of Environmental, Biological and Pharmaceutical Sciences and Technology, Via Vivaldi 43, 81100 Caserta, Italy; Interuniversity Centre for Research on Bioactive Peptides (CIRPEB), University of Naples Federico II, Via Mezzocannone 16, 80134 Naples, Italy. Electronic address: roberto.fattorusso@unina2.it.
Roberto
R
Fattorusso
D016960:Agrobacterium tumefaciens / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D003903:Deuterium / Q000737:chemistry; D006639:Histidine; D006859:Hydrogen / Q000737:chemistry; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D015032:Zinc / Q000737:chemistry / Q000378:metabolism*; D016335:Zinc Fingers / Q000235:genetics
D001426:Bacterial Proteins; D006639:Histidine; D006859:Hydrogen; D004247:DNA; D003903:Deuterium; D015032:Zinc
H/D exchange; Metal binding proteins; NMR; Prokaryotic zinc finger