Crystal structure of the zinc-, cobalt-, and iron-containing adenylate kinase from Desulfovibrio gigas: a novel metal-containing adenylate kinase from Gram-negative bacteria

Abstract

Adenylate kinases (AK) from Gram-negative bacteria are generally devoid of metal ions in their LID domain. However, three metal ions, zinc, cobalt, and iron, have been found in AK from Gram-negative bacteria. Crystal structures of substrate-free AK from Desulfovibrio gigas with three different metal ions (Zn 2? , Zn-AK; Co 2? , Co-AK; and Fe 2? , Fe-AK) bound in its LID domain have been determined by X-ray crystallography to resolutions 1.8, 2.0, and 3.0 Å , respectively. The zinc and iron forms of the enzyme were crystallized in space group I222, whereas the cobalt-form crystals were C2. The presence of the metals was confirmed by calculation of anomalous difference maps and by X-ray fluorescence scans. The work presented here is the first report of a structure of a metal-containing AK from a Gram-negative bacterium. The native enzyme was crystallized, and only zinc was detected in the LID domain. Co-AK and Fe-AK were obtained by overexpressing the protein in Escherichia coli. Zn-AK and Fe-AK crystallized as monomers in the asymmetric unit, whereas Co-AK crystallized as a dimer. Nevertheless, all three crystal structures are very similar to each other, with the same LID domain topology, the only change being the presence of the different metal atoms. In the absence of any substrate, the LID domain of all holoforms of AK was present in a fully open conformational state. Normal mode analysis was performed to predict fluctuations of the LID domain along the catalytic pathway.

Normal Mode Analysis as a Routine Part of a Structural Investigation

“…The greatest number of studies used NMA to characterize the general flexibility and domain movements of the protein under study [132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148]. The use of NMA in these studies runs from the simple to the sophisticated.…”

Mapping the Dynamics Landscape of Conformational Transitions in Enzyme: The Adenylate Kinase Case

“…References (73)(74)(75)(76)(77)(78)(79)(80)(81)(82)(83)(84)(85)(86)(87) appear in the Supporting Material.…”

Hypoxanthine-Guanine Phosphoribosyltransferase/adenylate Kinase From Zobellia galactanivorans: A Bifunctional Catalyst for the Synthesis of Nucleoside-5′-Mono-, Di- and Triphosphates

“…Regarding to the AMPK domain, the typical adenylate kinase motifs are present in Zg HGPRT/AMPK amino acid sequence ( Figure 5A ): (i) the P-loop (also known as Walker A motif), that adopts a specific loop shape allowing the accommodation of phosphate moiety from ATP, (ii) the AMP binding domain, which ensures that the adenine group from adenylate is selectively distinguished from other nitrogenous bases, and (iii) the LID domain, which displays high mobility allowing the active site isolation and thus generates a hydrophobic environment, avoiding the hydrolysis and promoting phosphotransferase activity ( Formoso et al, 2015 ; Figure 5A ). Moreover, AMPK proteins have a α/β structure formed by 5 β-sheet surrounded by several a-helices ( Mukhopadhyay et al, 2010 ; Figure 5B ). Like other AMPKs, Zg HGPRT/AMPK shows the typical core domain formed by the P-loop and a high conserved 12-residue AMPK “fingerprint,” including the G-F-P-R sequence that contribute to the protein folding and stabilization ( Figure 5B ; Mukhopadhyay et al, 2010 ).…”

“…Moreover, AMPK proteins have a α/β structure formed by 5 β-sheet surrounded by several a-helices ( Mukhopadhyay et al, 2010 ; Figure 5B ). Like other AMPKs, Zg HGPRT/AMPK shows the typical core domain formed by the P-loop and a high conserved 12-residue AMPK “fingerprint,” including the G-F-P-R sequence that contribute to the protein folding and stabilization ( Figure 5B ; Mukhopadhyay et al, 2010 ). As inferred from Figure 5B , ATP would be located between the core and LID domains, while AMP would be sandwiched between the core and the AMP binding site ( Figure 5B ).…”