Limulus polyphemus Hemocyanin: 10 Å Cryo-EM Structure, Sequence Analysis, Molecular Modelling and Rigid-body Fitting Reveal the Interfaces Between the Eight Hexamers

Martin, Andreas; Depoix, Frank; Stöhr, Michael; Meissner, Ulrich; Hagner-Holler, Silke; Hammouti, Kada; Burmester, Thorsten; Heyd, Jochen; Wriggers, Willy; Markl, Jürgen

doi:10.1016/j.jmb.2006.11.075

Cited by 49 publications

(68 citation statements)

References 50 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Thus, U. pusilla Hc can be defined as a tetramer of 1 × 6-meric building blocks, in contrast to the 4 × 6-meric Hc of Chelicerates where strong interactions exist between hexamers to build up the 2 × 6-meric half molecules. In the case of tarantula, scorpion and horseshoe crab Hcs the quaternary organization depends on a precise topology of the distribution of the various subunits within the oligomer (Lamy et al, 1981;Markl et al, 1981;Markl and Decker, 1992;Voit et al, 2000;Martin et al, 2007). In particular, linker subunits are responsible for the aggregation of the two 2 × 6-mers and for the highly hierarchical order of interactions (Voit et al, 2000).…”

Section: Resultsmentioning

confidence: 99%

“…It was shown by multivariate statistical analysis applied to computer aligned electron microscopic images that the 4 × 6 Hcs of A. australis and E. californicum are very similar (Bijholt et al, 1982) and fit the model earlier derived for the 4 × 6 half molecules of Limulus polyphemus. Such 4 × 6-mers are quasi-square-planar and comprise two 2 × 6-meric halves in an antiparallel arrangement that come in contact edge-to-edge, leaving a "deep cleft" between them (Martin et al, 2007). No high-resolution structure is available for the crustacean 4 × 6-mer but electron microscopy (EM) analysis revealed a completely different quaternary structure.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

The molecular heterogeneity of hemocyanin: Structural and functional properties of the 4×6-meric protein of Upogebia pusilla (Crustacea)

Paoli

Giomi

Hellmann³

et al. 2007

Gene

View full text Add to dashboard Cite

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

The molecular heterogeneity of hemocyanin: Structural and functional properties of the 4×6-meric protein of Upogebia pusilla (Crustacea)

Paoli

Giomi

Hellmann³

et al. 2007

Gene

View full text Add to dashboard Cite

“…Salt bridges between some conserved histidine and glutamate residues on the contact surface between monomers of Eurypelma californicum Hc (37) are proposed to be important. Possible mediators of allosteric signals have been recently described for L. polyphemus Hc (38). The deoxy protein is known to exhibit different conformations, which are evident in changes in the binding of small molecule allosteric effectors.…”

Section: Discussionmentioning

confidence: 99%

Molecular Basis of the Bohr Effect in Arthropod Hemocyanin

Hirota

Kawahara

Beltramini

et al. 2008

Journal of Biological Chemistry

View full text Add to dashboard Cite

Flash photolysis and K-edge x-ray absorption spectroscopy (XAS) were used to investigate the functional and structural effects of pH on the oxygen affinity of three homologous arthropod hemocyanins (Hcs). Flash photolysis measurements showed that the well-characterized pH dependence of oxygen affinity (Bohr effect) is attributable to changes in the oxygen binding rate constant, k on , rather than changes in k off . In parallel, coordination geometry of copper in Hc was evaluated as a function of pH by XAS. It was found that the geometry of copper in the oxygenated protein is unchanged at all pH values investigated, while significant changes were observed for the deoxygenated protein as a function of pH. The interpretation of these changes was based on previously described correlations between spectral lineshape and coordination geometry obtained for model compounds of known structure (Blackburn, N. J., Strange, R. W., Reedijk, J., Volbeda, A., Farooq, A., Karlin, K. D., and Zubieta, J. (1989) Inorg. Chem., 28, 1349 -1357). A pH-dependent change in the geometry of cuprous copper in the active site of deoxyHc, from pseudotetrahedral toward trigonal was assigned from the observed intensity dependence of the 1s 3 4p z transition in x-ray absorption near edge structure (XANES) spectra. The structural alteration correlated well with increase in oxygen affinity at alkaline pH determined in flash photolysis experiments. These results suggest that the oxygen binding rate in deoxyHc depends on the coordination geometry of Cu(I) and suggest a structural origin for the Bohr effect in arthropod Hcs. Hemocyanins (Hcs)3 are oxygen carrier and storage proteins found in molluscs and arthropods. Significant differences are observed between mollusc and arthropod Hcs in size of the functional units and in their tertiary and quaternary structures. Specifically, arthropod Hcs are structurally homogenous oligomeric proteins with a minimal functional subunit of 75 kDa. Under physiological conditions in the presence of calcium, the subunits are typically arranged in hexamers and dodecamers. Removal of Ca 2ϩ with EDTA at neutral pH causes dissociation of the dodecamer into hexamers, which can be dissociated into monomers at alkaline pH (1). The different aggregation states are related to modified oxygen binding properties (2). As generally observed for respiratory proteins, a fundamental physiological property of Hc is its competence to bind oxygen with different affinity in response to allosteric effectors including hydrogen ions.Detailed structural information concerning the active site of Hcs is derived mainly from a limited set of x-ray crystallographic investigations. The active site structures of the deoxy form have been described for two arthropod Hcs, Panulirus interruptus (3) and Limulus polyphemus (subunit II) (4). However, a comparison of the results is limited by the different solution conditions from which the proteins were crystallized. In both examples, each Cu(I) of the binuclear active site is coordinated by the ⑀-nitrog...

show abstract

“…The MM values obtained for solutions of unliganded GltS at low ionic strength (Table 1, lines 1-4) indicate that Ͼ90% of the protein is present as an (␣␤) 6 hexamer. GltS solutions that had been preincubated for up to 16 h with the L-Gln analog MetS (1 mM) and the GltS substrate 2-OG (1 mM), alone (not shown) or in combination, yielded similar R g and MM values (Table 1, lines [17][18][19][20]. On the contrary, preincubation with NADP ϩ either alone or in combination with MetS and 2-OG (Table 1, lines 21-24 and 13-16) resulted in a decrease of both R g and MM, suggesting a shift in the oligomeric equilibrium toward smaller particles.…”

Section: Saxs and Stoichiometry Of The Nadph-glts Complex In Solutionmentioning

confidence: 99%

“…The combined use of cryo-EM and SAXS allowed us to obtain an electron density map of the GltS (␣␤) 6 hexamer at a subnanometer resolution, which has been reached only recently, and only in a few cases, for cryo-EM-based structure determinations (17)(18)(19)(20)(21)(22). Moreover, we propose a homology model of ␤GltS, and, more importantly, that of the ␣␤ protomer.…”

mentioning

confidence: 99%

The Subnanometer Resolution Structure of the Glutamate Synthase 1.2-MDa Hexamer by Cryoelectron Microscopy and Its Oligomerization Behavior in Solution

Cottevieille

Larquet

Jonić

et al. 2008

Journal of Biological Chemistry

View full text Add to dashboard Cite

The three-dimensional structure of the hexameric (␣␤) 6 1.2-MDa complex formed by glutamate synthase has been determined at subnanometric resolution by combining cryoelectron microscopy, small angle x-ray scattering, and molecular modeling, providing for the first time a molecular model of this complex ironsulfur flavoprotein. In the hexameric species, interprotomeric ␣-␣ and ␣-␤ contacts are mediated by the C-terminal domain of the ␣ subunit, which is based on a ␤ helical fold so far unique to glutamate synthases. The ␣␤ protomer extracted from the hexameric model is fully consistent with it being the minimal catalytically active form of the enzyme. The structure clarifies the electron transfer pathway from the FAD cofactor on the ␤ subunit, to the FMN on the ␣ subunit, through the low potential [4Fe-4S] 1؉/2؉ centers on the ␤ subunit and the [3Fe-4S] 0/1؉ cluster on the ␣ subunit. The (␣␤) 6 hexamer exhibits a concentration-dependent equilibrium with ␣␤ monomers and (␣␤) 2 dimers, in solution, the hexamer being destabilized by high ionic strength and, to a lower extent, by the reaction product NADP ؉ . Hexamerization seems to decrease the catalytic efficiency of the ␣␤ protomer only 3-fold by increasing the K m values measured for L-Gln and 2-OG. However, it cannot be ruled out that the (␣␤) 6 hexamer acts as a scaffold for the assembly of multienzymatic complexes of nitrogen metabolism or that it provides a means to regulate the activity of the enzyme through an as yet unknown ligand.Glutamate synthases (GltS) 2 are complex iron-sulfur flavoproteins that catalyze the reductive transfer of the L-Gln amide group to the C2 carbon of 2-oxoglutarate (2-OG), yielding two molecules of L-glutamate (L-Glu). GltS are found in bacteria, yeast, and plants, where they form with glutamine synthetase an essential pathway for ammonia assimilation (1-3). Bacterial NADPH-dependent GltS (NADPH-GltS) is formed by one ␣ subunit (␣GltS) and one ␤ subunit (␤GltS) of 162 and 52 kDa, respectively, for the Azospirillum brasilense GltS. The ␣␤ protomer contains one FAD (on ␤GltS), one FMN (on ␣GltS), and three different iron-sulfur clusters (one [3Fe-4S] 0/1ϩ cluster on ␣GltS and two [4Fe-4S] 1ϩ/2ϩ centers on ␤GltS; Fig. 1A). On the basis of sequence, structural, and mechanistic similarities, the NADPH-GltS serves as a model for the other two main forms of GltS, namely (i) the ferredoxin-dependent GltS found in cyanobacteria and photosynthetic tissues of plants, which is similar to ␣GltS and (ii) the eukaryotic type of GltS, which is NADH-dependent and is found in yeast, nonphotosynthetic tissues of plants, and lower eukaryotes; this GltS species is formed by a single polypeptide chain derived from the fusion of bacterial ␣ and ␤ subunits.Several lines of evidence support an essential role of GltS in all of these cell types, making it a potential target of novel drugs. For example, the NADPH-GltS has been found to be essential in Mycobacterium tuberculosis, where it has been shown that Ͻ1 M azaserine, a GltS inhibitor, is sufficient t...

show abstract

Limulus polyphemus Hemocyanin: 10 Å Cryo-EM Structure, Sequence Analysis, Molecular Modelling and Rigid-body Fitting Reveal the Interfaces Between the Eight Hexamers

Cited by 49 publications

References 50 publications

The molecular heterogeneity of hemocyanin: Structural and functional properties of the 4×6-meric protein of Upogebia pusilla (Crustacea)

The molecular heterogeneity of hemocyanin: Structural and functional properties of the 4×6-meric protein of Upogebia pusilla (Crustacea)

Molecular Basis of the Bohr Effect in Arthropod Hemocyanin

The Subnanometer Resolution Structure of the Glutamate Synthase 1.2-MDa Hexamer by Cryoelectron Microscopy and Its Oligomerization Behavior in Solution

Contact Info

Product

Resources

About