Functional and structural roles of the N-terminal extension in Methanosarcina acetivorans protoglobin

Ciaccio, Chiara; Pesce, Alessandra; Tundo, Grazia R.; Tilleman, Lesley; Bertolacci, Laura; Dewilde, Sylvia; Moëns, Luc; Ascenzi, Paolo; Bolognesi, Martino; Nardini, M.; Coletta, Massimo

doi:10.1016/j.bbapap.2013.02.026

Cited by 11 publications

(8 citation statements)

References 24 publications

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“…The only major difference was the presence of the extra terminal extensions in AHb3, of which the C-terminus significantly protruded from the core domain (Figure ). A similar kind of N- and C-terminal extensions has previously been reported for some bacterial truncated hemoglobins (MtrHbN and HGbIV) and protoglobins, suggesting their evolutionary significance. − AHb3 shares seven conserved residues with the bacterial truncated Hbs in the heme pocket at topological positions: B9, B10, CD1, E7, E11, E14, and F8 (Figure ). Except HGbIV, residues lining the distal pocket of AHb3 were conserved across members of the trHbO group (Table ), with minor differences in their spatial orientation (Figure ), which may lead to variation in ligand binding regulation mechanisms.…”

Section: Resultssupporting

confidence: 70%

Structural and Functional Significance of the N- and C-Terminal Appendages in Arabidopsis Truncated Hemoglobin

et al. 2016

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Plant hemoglobins constitute three distinct groups: symbiotic, nonsymbiotic, and truncated hemoglobins. Structural investigation of symbiotic and nonsymbiotic (class I) hemoglobins revealed the presence of a vertebrate-like 3/3 globin fold in these proteins. In contrast, plant truncated hemoglobins are similar to bacterial truncated hemoglobins with a putative 2/2 α-helical globin fold. While multiple structures have been reported for plant hemoglobins of the first two categories, for plant truncated globins only one structure has been reported of late. Here, we report yet another crystal structure of the truncated hemoglobin from Arabidopsis thaliana (AHb3) with two water molecules in the heme pocket, of which one is distinctly coordinated to the heme iron, unlike the only available crystal structure of AHb3 with a hydroxyl ligand. AHb3 was monomeric in its crystallographic asymmetric unit; however, dimer was evident in the crystallographic symmetry, and the globin indeed existed as a stable dimer in solution. The tertiary structure of the protein exhibited a bacterial-like 2/2 α-helical globin fold with an additional N-terminal α-helical extension and disordered C-termini. To address the role of these extended termini in AHb3, which is yet unknown, N- and C-terminal deletion mutants were created and characterized and molecular dynamics simulations performed. The C-terminal deletion had an insignificant effect on most properties but perturbed the dimeric equilibrium of AHb3 and significantly influenced azide binding kinetics in the ferric state. These results along with the disordered nature of the C-terminus indicated its putative role in intramolecular or intermolecular interactions probably regulating protein-ligand and protein-protein interactions. While the N-terminal deletion did not change the overall globin fold, stability, or ligand binding kinetics, it seemed to have influenced coordination at the heme iron, the hydration status of the active site, and the quaternary structure of AHb3. Evidence indicated that the N-terminus is the predominant factor regulating the quaternary interaction appropriate to physiological requirements, dynamics of the side chains in the heme pocket, and tunnel organization in the protein matrix.

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Section: Resultssupporting

confidence: 70%

Structural and Functional Significance of the N- and C-Terminal Appendages in Arabidopsis Truncated Hemoglobin

et al. 2016

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“…The 20-residue N-terminal loop, together with other extended loops connecting the C-helix with the E-helix and the F-helix with the G-helix (which all are longer than in classical globins and conserved in Pgbs), completely bury the haem within the protein matrix, such that the haem propionates are solvent inaccessible (Figure S1). This structural feature, which is particularly unusual within the globin family structures, wherefore approximately 30% of the haem surface is normally solvent accessible, plays important role in ligand binding [11]. Thus, in Pgb the access of diatomic ligands, such as O 2 , CO, and NO, to the haem is granted by two orthogonal apolar tunnels that reach the haem distal region from entry sites at the B/G and B/E helix interfaces (Figure S1).…”

Section: Introductionmentioning

confidence: 96%

Structure and Haem-Distal Site Plasticity in Methanosarcina acetivorans Protoglobin

et al. 2013

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Protoglobin from Methanosarcina acetivorans C2A (MaPgb), a strictly anaerobic methanogenic Archaea, is a dimeric haem-protein whose biological role is still unknown. As other globins, protoglobin can bind O2, CO and NO reversibly in vitro, but it displays specific functional and structural properties within members of the hemoglobin superfamily. CO binding to and dissociation from the haem occurs through biphasic kinetics, which arise from binding to (and dissociation from) two distinct tertiary states in a ligation-dependent equilibrium. From the structural viewpoint, protoglobin-specific loops and a N-terminal extension of 20 residues completely bury the haem within the protein matrix. Thus, access of small ligand molecules to the haem is granted by two apolar tunnels, not common to other globins, which reach the haem distal site from locations at the B/G and B/E helix interfaces. Here, the roles played by residues Trp(60)B9, Tyr(61)B10 and Phe(93)E11 in ligand recognition and stabilization are analyzed, through crystallographic investigations on the ferric protein and on selected mutants. Specifically, protein structures are reported for protoglobin complexes with cyanide, with azide (also in the presence of Xenon), and with more bulky ligands, such as imidazole and nicotinamide. Values of the rate constant for cyanide dissociation from ferric MaPgb-cyanide complexes have been correlated to hydrogen bonds provided by Trp(60)B9 and Tyr(61)B10 that stabilize the haem-Fe(III)-bound cyanide. We show that protoglobin can strikingly reshape, in a ligand-dependent way, the haem distal site, where Phe(93)E11 acts as ligand sensor and controls accessibility to the haem through the tunnel system by modifying the conformation of Trp(60)B9.

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“…Although other vertebrate globins (Mb, Hb, Ngb) lack terminal extensions, similar appendages have been observed in several bacterial globins, including truncated hemoglobins from M. tuberculosis (Savard et al 2011), A. thaliana (Mukhi et al 2016), and M. infernorum (Jamil et al 2014), as well as in protoglobin from M. acetivorans (Ciaccio et al 2013). Interestingly, crystal structure of truncated hemoglobin from A. thaliana (AHb3) exhibits identical secondary structure of terminal fragments as observed in Cygb (Mukhi et al 2016;Reeder and Hough 2014).…”

Section: Background and Significancesupporting

confidence: 52%

Structure-Function Relationships in Hexacoordinate Heme Proteins: Mechanism of Cytoglobin Interactions with Exogenous Ligands

Tangar¹

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Functional and structural roles of the N-terminal extension in Methanosarcina acetivorans protoglobin

Cited by 11 publications

References 24 publications

Structural and Functional Significance of the N- and C-Terminal Appendages in Arabidopsis Truncated Hemoglobin

Structural and Functional Significance of the N- and C-Terminal Appendages in Arabidopsis Truncated Hemoglobin

Structure and Haem-Distal Site Plasticity in Methanosarcina acetivorans Protoglobin

Structure-Function Relationships in Hexacoordinate Heme Proteins: Mechanism of Cytoglobin Interactions with Exogenous Ligands

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