Structural Determinants in the Group III Truncated Hemoglobin from Campylobacter jejuni

Truncated hemoglobins (trHbs) constitute a distinct lineage in the globin superfamily, distantly related in size and fold to myoglobin and monomeric hemoglobins. Their phylogenetic analyses revealed that three groups (I, II, and III) compose the trHb family. Group I and II trHbs adopt a simplified globin fold, essentially composed of a 2-on-2 α-helical sandwich, wrapped around the heme group. So far no structural data have been reported for group III trHbs. Here we report the three-dimensional structure of the group III trHbP from the eubacterium Campylobacter jejuni. The 2.15-Å resolution crystal structure of C. jejuni trHbP (cyano-met form) shows that the 2-on-2 trHb fold is substantially conserved in the trHb group III, despite the absence of the Gly-based sequence motifs that were considered necessary for the attainment of the trHb specific fold. The heme crevice presents important structural modifications in the C-E region and in the FG helical hinge, with novel surface clefts at the proximal heme site. Contrary to what has been observed for group I and II trHbs, no protein matrix tunnel/cavity system is evident in C. jejuni trHbP. A gating movement of His(E7) side chain (found in two alternate conformations in the crystal structure) may be instrumental for ligand entry to the heme distal site. Sequence conservation allows extrapolating part of the structural results here reported to the whole trHb group III. Truncated hemoglobins (trHbs) constitute a distinct lineage in the globin superfamily, distantly related in size and fold to myoglobin and monomeric hemoglobins. Their phylogenetic analyses revealed that three groups (I, II, and III) compose the trHb family. Group I and II trHbs adopt a simplified globin fold, essentially composed of a 2-on-2 α-helical sandwich, wrapped around the heme group. So far no structural data have been reported for group III trHbs. Here we report the three-dimensional structure of the group III trHbP from the eubacterium Campylobacter jejuni. The 2.15-Å resolution crystal structure of C. jejuni trHbP (cyano-met form) shows that the 2-on-2 trHb fold is substantially conserved in the trHb group III, despite the absence of the Gly-based sequence motifs that were considered necessary for the attainment of the trHb specific fold. The heme crevice presents important structural modifications in the C-E region and in the FG helical hinge, with novel surface clefts at the proximal heme site. Contrary to what has been observed for group I and II trHbs, no protein matrix tunnel/cavity system is evident in C. jejuni trHbP. A gating movement of His(E7) side chain (found in two alternate conformations in the crystal structure) may be instrumental for ligand entry to the heme distal site. Sequence conservation allows extrapolating part of the structural results here reported to the whole trHb group III. Recent phylogeny of the globin superfamily indicates that it contains three lineages belonging to two structural classes: one showing a 3-on-3 α-helical sandwich (animal, plant, and chimeric globins) and one having a 2-on-2 α-helical sandwich (truncated hemoglobins: trHbs). 2The abbreviations used are: trHb, truncated hemoglobin; Hb, hemoglobin; Mb, myoglobin; Mt-trHbN, Mycobacterium tuberculosis trHbN; Mt-trHbO, Mycobacterium tuberculosis trHbO; Bs-trHbO, Bacillus subtilis trHbO; Cj-trHbP, Campylobacter jejuni trHbP; r.m.s., root mean square. Although no definitive conclusion can be drawn about the ancestral state of the globin fold, the occurrence of the 2-on-2-fold, but not of an isolated 3-on-3 fold, in all three kingdoms of life suggests that the 2-on-2 fold is the ancestral fold. On the other hand, functional studies indicate that the predominant function of globins would be enzymatic, O2 transport and storage being a specialized function associated with the evolution of metazoans (1Vinogradov S.N. Hoogewijs D. Bailly X. Arredondo-Peter R. Guertin M. Giugh J. Dewilde S. Moens L. Vanfleteren J.R. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 11385-11389Crossref PubMed Scopus (132) Google Scholar). TrHbs are widely distributed in eubacteria and plants and are found in some unicellular eukaryotes. They are distantly related to the 3-on-3 globins, showing less than 20% overall identity with the latter (2Wittenberg J.B. Bolognesi M. Wittenberg B.A. Guertin M. J. Biol. Chem. 2002; 277: 871-874Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar). Phylogenetic analysis based on protein sequences shows that trHbs branch into three groups, designated I, II, and III (2Wittenberg J.B. Bolognesi M. Wittenberg B.A. Guertin M. J. Biol. Chem. 2002; 277: 871-874Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar, 3Vuletich D.A. Lecomte J.T. J. Mol. Evol. 2006; 62: 196-210Crossref PubMed Scopus (105) Google Scholar), whose members are distinguished by the N, O, and P suffixes, respectively. Group I and group II trHbs further separate into two and four subgroups, respectively, whereas group III trHbs display a high level of overall conservation. Sequence identity between trHbs from different groups is low (≤20% overall identity), but may be higher than 80% within a given group. TrHbs are largely present in eubacteria, with some bacteria displaying more than one trHb belonging to different groups. Analysis of the distribution of trHbs suggested a scenario for the evolution of the different groups where the group II trHb gene is the ancestral gene, and group I and group III genes are the results of duplications and transfer events (3Vuletich D.A. Lecomte J.T. J. Mol. Evol. 2006; 62: 196-210Crossref PubMed Scopus (105) Google Scholar). At present, four trHbs from group I and two from group II have been structurally characterized (4Pesce A. Couture M. Dewilde S. Guertin M. Yamauchi K. Ascenzi P. Moens L. Bolognesi M. EMBO J. 2000; 19: 2424-2434Crossref PubMed Google Scholar, 9Giangiacomo L. Ilari A. Boffi A. Morea V. Chiancone E. J. Biol. Chem. 2005; 280: 9192-9202Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). These studies revealed that specific residue deletions and substitutions distributed throughout the trHb sequence allow the achievement of the simplified fold, whereas granting enough affinity for the heme, and ligand access to the heme-iron atom. Among sequence peculiarities, two Gly-Gly sequence motifs have been deemed necessary for stabilization of the trHb fold in groups I and II, and identified in their related amino acid sequences (3Vuletich D.A. Lecomte J.T. J. Mol. Evol. 2006; 62: 196-210Crossref PubMed Scopus (105) Google Scholar, A. Couture M. Dewilde S. Guertin M. Yamauchi K. Ascenzi P. Moens L. Bolognesi M. EMBO J. 2000; 19: 2424-2434Crossref PubMed Google Scholar). studies in to and globins, the distal heme of trHbs may between groups and between for the acid have been their the sequence and the within the globin has been for other the heme distal are not conserved in trHbs. The proximal the heme is the in trHbs (2Wittenberg J.B. Bolognesi M. Wittenberg B.A. Guertin M. J. Biol. Chem. 2002; 277: 871-874Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar, 3Vuletich D.A. Lecomte J.T. J. Mol. Evol. 2006; 62: 196-210Crossref PubMed Scopus (105) Google Scholar). trHbs far with affinity from to TrHbs from Mycobacterium tuberculosis and Campylobacter jejuni have been for their with Mycobacterium that of the gene the of to from that may a in M. tuberculosis from in functional is by the that the of into with a of C. M. Wittenberg Wittenberg J. Guertin M. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar). has been to R. Mol. 2002; PubMed Scopus Google Scholar). On the other hand, a trHbP C. jejuni found with to high and O2 at the by not to has been that may a in 2005; PubMed Scopus Google Scholar, 2006; PubMed Scopus Google Scholar). of the group III trHb amino acid sequences indicates the absence of the Gly-Gly and a of specific residue substitutions that on the for groups I and On the other hand, analysis of group III trHb amino acid sequences indicates a high level of residue conservation within higher than within group I and group II trHbs (3Vuletich D.A. Lecomte J.T. J. Mol. Evol. 2006; 62: 196-210Crossref PubMed Scopus (105) Google Scholar). on the the for structural of a group III trHb has substantially in the in of the sequence conservation reported it would allow of structural to of the group III trHb C. jejuni is a is the predominant in two distinct hemoglobins by the and for a group III trHb, and a 2005; PubMed Scopus Google Scholar, 2006; PubMed Scopus Google Scholar). with on structure to function in the trHb we present here the results of a on C. jejuni trHbP in at 2.15-Å that the of the trHb fold in C. jejuni trHbP are than to observed for group II of group III trHbs are an the of specific and the absence of a ligand protein matrix characterized in group I, and in group II trHbs. crystal structure suggests that ligand entry into the heme distal is by the M. D. M. C. Ascenzi P. Mol. Biol. PubMed Scopus Google Scholar). and of from C. jejuni by J. from of used the to the region of The used were and The into the and the protein in E. were at in of of and of the at at were for an The were by at for at and conserved at The with of and I The were by a at at were by at for at The with The at for in and the with at in The a of of and the protein with a of in at with a were and by The protein in of and the at The protein a with and at with a were and by and The protein of the the protein a with studies the by the protein with The protein a and with a of in and at with a were and by and were at The protein by The heme identified and by the and of C. jejuni trHbP the The protein at a at K. within a They were in a and to the with 20% to and data The to 2.15-Å and to the group with a The is with two trHbP of in about having of a at K. The were in a were to the with 20% to data These to resolution and were characterized belonging to the group with a The is with two trHbP data were and and of Scholar, of the on and Scholar). are reported in for overall in a and structure by the A. A. J. Scopus Google in the Biol. PubMed Scopus Google Scholar). The crystal structure of truncated group II from Bacillus subtilis L. Ilari A. Boffi A. Morea V. Chiancone E. J. Biol. Chem. 2005; 280: 9192-9202Abstract Full Text Full Text PDF PubMed Scopus (63) Google used with side truncated to in of between the two amino acid The and in the resolution a for a the group in of of all the and the heme group in the and were and 2.15-Å of the M. Biol. PubMed Scopus Google Scholar), and the Biol. PubMed Scopus Google and were to the and the protein that were not in the At the of the and were of the The and and where and are the observed and structure is on of the were not used the of of heme of of of of from in in where and are the observed and structure is on of the were not used the in a The structure of used a to the structure and the crystal the At the of the were of the The and The J. Google and J. Mol. PubMed Scopus Google were used to the of the protein and the protein matrix The E. K. R. K. of in used to within the crystal and structure have been with the J. 2000; PubMed Scopus Google Scholar), with entry and respectively. of the structure of on belonging to the group to A and by of based on the of group II from subtilis L. Ilari A. Boffi A. Morea V. Chiancone E. J. Biol. Chem. 2005; 280: 9192-9202Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). The structure at 2.15-Å to and of and respectively, with a in chain in chain no is observed for the two of of the two the a of results to A and of the Cj-trHbP, in the group were and a resolution data and of and respectively, with of of the two a of of the from the and crystal in the for the on the shows the of the 2-on-2 α-helical (2Wittenberg J.B. Bolognesi M. Wittenberg B.A. Guertin M. J. Biol. Chem. 2002; 277: 871-874Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar, A. Couture M. Dewilde S. Guertin M. Yamauchi K. Ascenzi P. Moens L. Bolognesi M. EMBO J. 2000; 19: 2424-2434Crossref PubMed Google Scholar, 9Giangiacomo L. Ilari A. Boffi A. Morea V. Chiancone E. J. Biol. Chem. 2005; 280: 9192-9202Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), a of the globin 3-on-3 α-helical sandwich PubMed Scopus Google Scholar, L. C. PubMed Scopus Google and to of the and of with the of group I and II trHbs (4Pesce A. Couture M. Dewilde S. Guertin M. Yamauchi K. Ascenzi P. Moens L. Bolognesi M. EMBO J. 2000; 19: 2424-2434Crossref PubMed Google Scholar, M. A. Ascenzi P. Guertin M. Bolognesi M. EMBO J. PubMed Scopus Google Scholar, M. Ascenzi P. Guertin M. Bolognesi M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, 9Giangiacomo L. Ilari A. Boffi A. Morea V. Chiancone E. J. Biol. Chem. 2005; 280: 9192-9202Abstract Full Text Full Text PDF PubMed Scopus (63) Google in the the evident in the amino acid sequences of the whole trHb the structural analysis the conservation of the 2-on-2 trHb fold, but it some structural of group III Among A is in Cj-trHbP, and the in an in the to the region in group I and II trHbs and of the region is by between O, and being structurally related to the absence of Gly-Gly sequence motifs at the and of group III trHbs that the observed of the region is by three in an conformations to region be Lecomte J.T. J. Mol. Biol. 2002; PubMed Scopus Google Scholar). The Gly-based motifs are and are sequence in group I and II trHbs (2Wittenberg J.B. Bolognesi M. Wittenberg B.A. Guertin M. J. Biol. Chem. 2002; 277: 871-874Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar, 3Vuletich D.A. Lecomte J.T. J. Mol. Evol. 2006; 62: 196-210Crossref PubMed Scopus (105) Google Scholar), where the A in a and (4Pesce A. Couture M. Dewilde S. Guertin M. Yamauchi K. Ascenzi P. Moens L. Bolognesi M. EMBO J. 2000; 19: 2424-2434Crossref PubMed Google Scholar, M. A. Ascenzi P. Guertin M. Bolognesi M. EMBO J. PubMed Scopus Google Scholar). a is the residue of the Gly-Gly in the is whose side chain the by the side chain of in group I and II trHbs between the and are by an distribution of at the helical the absence of the Gly-Gly at the of not the to the the the protein the to the A structural of group III trHbs is a acid between the and of the region in not the of to the heme distal site. it the of the and to the in group I and II trHb one and one and the protein structure in the region between the and to in the three trHb groups. in the a α-helical with the helical not by group I and II trHbs but by globins M. D. M. C. Ascenzi P. Mol. Biol. PubMed Scopus Google Scholar). of the and is by a the group III conserved to and to the side chain is from the region by the of structural in the group III trHb fold are the and FG suggested by their amino acid sequences from of group I and II trHbs the not the α-helical found in group II trHbs, and is different from that of group I (4Pesce A. Couture M. Dewilde S. Guertin M. Yamauchi K. Ascenzi P. Moens L. Bolognesi M. EMBO J. 2000; 19: 2424-2434Crossref PubMed Google Scholar), and Lecomte J.T. J. Mol. Biol. 2002; PubMed Scopus Google Scholar, J.T. S. J. Mol. Biol. PubMed Scopus Google Scholar), being more to M. tuberculosis M. A. Ascenzi P. Guertin M. Bolognesi M. EMBO J. PubMed Scopus Google and The FG structure in is by the of three These the of the hinge, having a the of the of the and between the and in is in it not of group I trHbs M. A. M. Dewilde S. A. Ascenzi P. Guertin M. Moens L. Wittenberg J.B. Bolognesi M. J. 2005; PubMed Scopus Google Scholar), found in group II M. Ascenzi P. Guertin M. Bolognesi M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google and L. Ilari A. Boffi A. Morea V. Chiancone E. J. Biol. Chem. 2005; 280: 9192-9202Abstract Full Text Full Text PDF PubMed Scopus (63) Google and Contrary to what has been observed in group I and II trHbs (2Wittenberg J.B. Bolognesi M. Wittenberg B.A. Guertin M. J. Biol. Chem. 2002; 277: 871-874Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar, M. A. Dewilde S. J. Ascenzi P. Guertin M. Bolognesi M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), of the overall structure shows no evident protein matrix tunnel/cavity by a a is used two can be is by by and the other is by and being between the and the heme distal site. the a that a between the heme distal and The in in the region the protein matrix branch in group I and II trHbs, but it is not to the heme distal to the of and are present in the of and crystal the found in different crystal display different and the has a of to of the surface of is by the and E. K. R. K. of in Scholar). The of to of the not specific that the of a that the protein a 2006; PubMed Scopus Google Scholar). A. M. C. A. P. M. and M. results that the observed are by the high protein than being functional and the of the indicates that in the heme group is by the to the of globins PubMed Scopus Google Scholar). of the of the heme can be related to the specific of the and to the heme and to specific at and found for group II M. Ascenzi P. Guertin M. Bolognesi M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google and for L. Ilari A. Boffi A. Morea V. Chiancone E. J. Biol. Chem. 2005; 280: 9192-9202Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). of the heme in is to the proximal of the heme and with protein A to is by the heme at from A and in whereas it a to in chain between the two is related to of the around the heme by different conformations of the distal is conserved in all three trHb groups, whereas at is in group II trHbs (2Wittenberg J.B. Bolognesi M. Wittenberg B.A. Guertin M. J. Biol. Chem. 2002; 277: 871-874Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar, 3Vuletich D.A. Lecomte J.T. J. Mol. Evol. 2006; 62: 196-210Crossref PubMed Scopus (105) Google Scholar). is to is conserved in group III trHb A heme is present in group I from M. tuberculosis (3Vuletich D.A. Lecomte J.T. J. Mol. Evol. 2006; 62: 196-210Crossref PubMed Scopus (105) Google and from J.T. S. J. Mol. Biol. PubMed Scopus Google Scholar), and in group II from M. tuberculosis M. Ascenzi P. Guertin M. Bolognesi M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google and subtilis L. Ilari A. Boffi A. Morea V. Chiancone E. J. Biol. Chem. 2005; 280: 9192-9202Abstract Full Text Full Text PDF PubMed Scopus (63) Google The of the heme in the proximal region is by and all of the The and of the proximal is of an that the heme of the Analysis of the the heme indicates a the PubMed Scopus Google Scholar), with the in a to the heme of the proximal is in with the observed observed 2006; PubMed Scopus Google Scholar). The side of the proximal heme is largely by being to the heme from the The from group II and Bs-trHbO, where the heme proximal residue is to an in a to the residue L. Ilari A. Boffi A. Morea V. Chiancone E. J. Biol. Chem. 2005; 280: 9192-9202Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Analysis of surface shows the of two surface around the heme proximal one of is between the and the the FG The crevice between the FG and the of the to the heme from the being from the proximal by residue by and by respectively. of the protein surface shows that the region by the and the the heme and essentially by of residue is the three trHb groups at at the of a low surface can the proximal heme to to the heme distal and the ligand in of are conserved in more than one trHb with the of is conserved in group III the distal a in ligand stabilization by group to the The ligand is to the heme with a of ligand stabilization is by the of is at from the reported have been the two protein At the of the distal conserved in trHb groups II and III, the part of the heme distal the of from the site. is and in with the heme at the and A and The is of that found in group II L. Ilari A. Boffi A. Morea V. Chiancone E. J. Biol. Chem. 2005; 280: 9192-9202Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), with residue for and the found and the matrix the by the in but it is not in with the to what is observed for the L. Ilari A. Boffi A. Morea V. Chiancone E. J. Biol. Chem. 2005; 280: 9192-9202Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). with group I trHbs, the that found in M. Guertin M. Boffi A. A. M. Bolognesi M. Ascenzi P. PubMed Scopus Google Scholar), with to substitutions in the latter protein of and at and respectively. the side chain the and to the of the observed in Cj-trHbP, residue would be to the is part of the the within the globin and to the heme distal with residue in the part of the a of within the is observed in for the to all globin and a conserved residue in all three trHb groups, a at the of the distal with the heme of is on the surface of the protein and may access to the distal to the is to access to the distal the analysis of the surface a shows that is a crevice between the and side and the heme A. with the crevice four and three in A and respectively. in chain is present in two alternate and to to the side chain the to the heme distal A and The is by a between the and O, whereas a to the is in chain A by the of a for the of the the is not in with protein with the being more than from the group and the a distal from the stabilization observed in Mb, where the His(E7) is in the in the heme distal M. C. R. A. M. Wittenberg J.B. Boffi A. Ascenzi P. J. Full Text Full Text PDF PubMed Scopus Google Scholar). can be related in to the of in an to the heme than in a the proximal residue and the heme group of and Cj-trHbP, the His(E7) of the two the heme distal residue of being from the heme, with no to with the A of the His(E7) residue is observed is with group I and II for to the the distal about and from the heme to the in C. and M. tuberculosis Sequence Analysis in trHb Group sequence identity in the to other members of trHb group the is whose sequence is about to that of of amino acid sequence based on the present crystal structure allows conserved at structural The of conserved to be higher in group III than in group I II trHbs, by and Lecomte (3Vuletich D.A. Lecomte J.T. J. Mol. Evol. 2006; 62: 196-210Crossref PubMed Scopus (105) Google based on their sequence Cj-trHbP, the three-dimensional structure to be reported for group III trHbs, to be a structural for the whole group III. Analysis of sequences in of the three-dimensional structure shows that a level of sequence is within the structure associated to the proximal and distal heme in heme stabilization a conservation of the distal the conservation of and may for into ligand of Cj-trHbP, to group II trHbs, the ligand is by to and to the is that a distal based on distal is a structural of trHbs (2Wittenberg J.B. Bolognesi M. Wittenberg B.A. Guertin M. J. Biol. Chem. 2002; 277: 871-874Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar, A. Couture M. Dewilde S. Guertin M. Yamauchi K. Ascenzi P. Moens L. Bolognesi M. EMBO J. 2000; 19: 2424-2434Crossref PubMed Google Scholar, 9Giangiacomo L. Ilari A. Boffi A. Morea V. Chiancone E. J. Biol. Chem. 2005; 280: 9192-9202Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, M. A. M. Dewilde S. A. Ascenzi P. Guertin M. Moens L. Wittenberg J.B. Bolognesi M. J. 2005; PubMed Scopus Google Scholar). conservation around the heme is evident for and on the proximal and to in heme stabilization and and the be that despite being in stabilization of the heme in Cj-trHbP, is not conserved in trHb group III, whereas at is in group II trHbs, and a heme M. A. Ascenzi P. Guertin M. Bolognesi M. EMBO J. PubMed Scopus Google Scholar). A low level of sequence conservation is found for the two that may access to the heme from the proximal of the reported sequence proximal access are to be a structural to The and sequence analyses on group I and group II trHbs two Gly-Gly sequence at the hinge, and to the respectively, of the trHb fold. one conserved residue identified of the Gly-Gly (3Vuletich D.A. Lecomte J.T. J. Mol. Evol. 2006; 62: 196-210Crossref PubMed Scopus (105) Google Scholar, M. A. Ascenzi P. Guertin M. Bolognesi M. EMBO J. PubMed Scopus Google Scholar). of the structure and group III trHb sequences shows that the residue is conserved in group III trHb, and an four is conserved in group III, but not in group I and II trHbs. On the an evident of group III trHbs is the absence of the two and Gly-Gly on the results here for the it be that the latter motifs are not to the of the 2-on-2 trHb fold. The absence of Gly-Gly motifs in group III trHbs by a structure of the in and with of the protein matrix observed in group I trHbs. side chain substitutions at the in group I and II trHbs are evident and conserved within group III members at at where a group III for the residue present in group I and at and in the structural analysis of allows to that the protein tunnel/cavity system for group I trHbs M. A. Ascenzi P. Guertin M. Bolognesi M. EMBO J. PubMed Scopus Google Scholar, M. A. M. Dewilde S. A. Ascenzi P. Guertin M. Moens L. Wittenberg J.B. Bolognesi M. J. 2005; PubMed Scopus Google is not to be a structural conserved in group III trHbs. On the other hand, the of a sequence between the and and a sequence at and that the of the distal and ligand gating for Cj-trHbP, may be a of group III trHbs. The three-dimensional structure of the on the fold and ligand of group III trHbs. the low sequence identity to trHbs from groups I and II, the structure based on a 2-on-2 α-helical sandwich is in group III the absence of the two Gly-Gly conserved at the and in group I and II trHbs, shows that motifs are for the achievement and stabilization of the trHb fold. Their absence is by evident structural at the where A is and the in a to that observed in the group I and II and at the hinge, whose not that of group I trHbs, the α-helical found in group II trHbs.

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