Quaternary structure of Artemia haemoglobin II: analysis of T and C polymer alignment and interpolymer interface

BACKGROUND: The brine shrimp Artemia expresses four different types of haemoglobin subunits namely C1, C2, T1 and T2. Two of these four subunits dimerize in different combinations to produce the three isoforms of the heterodimeric Artemia haemoglobin: HbI (C1 and C2), HbII (C1 and T2) and HbIII (T1 and T2). Previous biochemical, biophysical and computational analyses demonstrate that the T and C polymers are rings of nine concatenated globin domains, which are covalently joined by interdomain linkers. Two such rings stacked coaxially give the functional molecule. This research aimed to construct a quaternary structural model of Artemia HbII that shows the interpolymer interface and domain-domain alignment, using the MS3D (mass spectrometry for three dimensional analysis) approach. This involved introducing chemical crosslinks between the two polymers, cleaving with trypsin and analyzing the resulting products by mass spectrometry. This was followed by computational analysis of the mass spectrometry data using the program SearchXlinks to identify putatively crosslinked peptides. RESULTS: Six putative EGS (ethylene glycol bis [succinimidylsuccinate]) crosslinked tryptic peptides were identified. All of them support a model in which the EF helices of all domains are in contact along the interpolymer surface, and Domain 1 of the T-polymer aligns with Domain 1 of the C-polymer. Any two adjacent interpolymer domain pairs contact through the early Helix H and early Helix A. The orientation of domains is different from the subunit proposed model proposed previously by this group. Crosslinking with GMBS (N- [γ-maleimidobutyryloxy]succinimide ester) was also performed, and the results show good agreement with this model. CONCLUSION: The interpolymer EF-contact allows the hydrophobic E and F helices to be buried in the interface and therefore allow the complex to solubilize readily to facilitate efficient oxygen transport. Furthermore the EF-contact is a common contact in cooperative haemoglobins and thus the model is consistent with the cooperative behaviour of Artemia HbII

The front and top views of a section of the proposed EF: EF Domain 1: Domain 1 model of HbII complex

<p><b>Copyright information:</b></p><p>Taken from "Quaternary structure of haemoglobin II: analysis of T and C polymer alignment and interpolymer interface"</p><p>http://www.biomedcentral.com/1472-6807/7/26</p><p>BMC Structural Biology 2007;7():26-26.</p><p>Published online 18 Apr 2007</p><p>PMCID:PMC1865544.</p><p></p> The EF: EF Domain 1: Domain 1 model of HbII complex is shown in full in (front view), (top view) and (side view). The model was constructed using the software SPDBV37. Colour Codes: Helix A, grey; AB loop and helix B; green; Helix C, red; CD loop and helix D, purple; DE loop and helix E, cyan; EF loop and Helix F, blue; FG loop and helix G, yellow; GH loop and helix H; salmon

Inter-lysine (or lysine-cysteine) distances (in Å) residues which were experimentally shown to be crosslinked

<p><b>Copyright information:</b></p><p>Taken from "Quaternary structure of haemoglobin II: analysis of T and C polymer alignment and interpolymer interface"</p><p>http://www.biomedcentral.com/1472-6807/7/26</p><p>BMC Structural Biology 2007;7():26-26.</p><p>Published online 18 Apr 2007</p><p>PMCID:PMC1865544.</p><p></p> The TP and CP superscripts indicate T or C polymer residues. Note that this figure is the same as Figure 5a except that only crosslinked residues are shown

Reverse-phase high-performance liquid chromatography (RP-HPLC) chromatogram of completely trypsinolyzed HbII complex

<p><b>Copyright information:</b></p><p>Taken from "Quaternary structure of haemoglobin II: analysis of T and C polymer alignment and interpolymer interface"</p><p>http://www.biomedcentral.com/1472-6807/7/26</p><p>BMC Structural Biology 2007;7():26-26.</p><p>Published online 18 Apr 2007</p><p>PMCID:PMC1865544.</p><p></p> ESI-MS peaks subjected to ESI-MS zoom scan and tandem MS analyses and their corresponding RP-HPLC fractions were indicated on this Figure. Charge-states of peaks (inferred based on the ESI-MS zoom scan data) were indicated as superscripts (see also Figure 3b inset)

Unexpected intron location in non-vertebrate globin genes

The Caenorhabditis elegans and Artemia T4 globin sequences are highly homologous with other invertebrate globins.The intron/exon patterns of their genes display a single intron in the E and G helices respectively. Precoding introns in multirepeat globins are inserted in homologous positions. Comparison of the intron/exon patterns in the known globin gene sequences demonstrates that they are more diverse than first expected but nevertheless can be derived from an ancestral pattern having 3 introns and 4 exons

Alignement of 700 globin sequences: extent of amino acid substitution and its correlation with variation in volume

Seven-hundred globin sequences, including 146 nonvertebrate sequences, were aligned on the basis of conservation of secondary structure and the avoidance of gap penalties. Of the 182 positions needed to accommodate all the globin sequences, only 84 are common to all, including the absolutely conserved PheCD1 and HisF8. The mean number of amino acid substitutions per position ranges from 8 to 13 for all globins and 5 to 9 for internal positions. Although the total sequence volumes have a variation similar to 2-3%, the variation in volume per position ranges from similar to 13% for the internal to similar to 21% for the surface positions. Plausible correlations exist between amino acid substitution and the variation in volume per position for the 84 common and the internal but not the surface positions. The amino acid substitution matrix derived from the 84 common positions was used to evaluate sequence similarity within the globins and between the globins and phycocyanins C and colicins A, via calculation of pairwise similarity scores. The scores for globin-globin comparisons over the 84 common positions overlap the globin-phycocyanin and globin-colicin scores, with the former being intermediate. For the subset of internal positions, overlap is minimal between the three groups of scores. These results imply a continuum of amino acid sequences able to assume the common three-on-three alpha-helical structure and suggest that the determinants of the latter include sites other than those inaccessible to solvent