Quaternary Structure of the European Spiny Lobster (Palinurus elephas) 1×6-mer Hemocyanin from cryoEM and Amino Acid Sequence Data

Abstract

Arthropod hemocyanins are large respiratory proteins that are composed of up to 48 subunits (8 £ 6-mer) in the 75 kDa range. A 3D reconstruction of the 1 £ 6-mer hemocyanin from the European spiny lobster Palinurus elephas has been performed from 9970 single particles using cryoelectron microscopy. An 8 Å resolution of the hemocyanin 3D reconstruction has been obtained from about 600 final class averages. Visualisation of structural elements such as a-helices has been achieved. An amino acid sequence alignment shows the high sequence identity (. 80%) of the hemocyanin subunits from the European spiny lobster P. elephas and the American spiny lobster Panulirus interruptus. Comparison of the P. elephas hemocyanin electron microscopy (EM) density map with the known P. interruptus X-ray structure shows a close structural correlation, demonstrating the reliability of both methods for reconstructing proteins. By molecular modelling, we have found the putative locations for the amino acid sequence (597 -605) and the C-terminal end (654 -657), which are absent in the available P. interruptus X-ray data.

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

“…The resolution of the final cryo-EM structure of the 8×6mer is almost the same as in our previous 3D reconstruction of the native 1×6mer hemocyanin from P. elephas. 39 Indeed, the eight hexamers are clearly defined and exhibit molecular detail; notably, the 48 individual subunits are discernible ( Figure 3(a)-(c)). The handedness of the 1×6mer was confirmed by X-ray analysis of P. interruptus hemocyanin crystals; 4 this allowed unambiguous assignment of the correct handedness to the cryo-EM density map of the 8×6mer.…”

“…This 3D reconstruction allowed a convincing fit with the crystal structure of Panulirus interruptus 1×6mer, and molecular modelling of some parts. 39 This result encouraged us to study, by cryo-EM, the 8×6mer hemocyanin of L. polyphemus.…”

Structural characterisation of theN-glycan moiety of the barnacle settlement-inducing protein complex (SIPC)

“…TEM images revealed both circular shapes of 14nm diameter and rectangular structures of 16ϫ9nm (Rousselot et al, 2006). Again focusing on haemocyanin, Meissner and colleagues presented the structure for the haemocyanin of the European spiny lobster Palinurus elephas, using a combination of cryoelectron microscopy and analysis of the amino acid sequence (Meissner et al, 2003). Comparison of the sequence with that of the American counterpart, Panulirus interruptus, showed more than 80% sequence homology.…”

Structure and Characterization of Eriphia verrucosa Hemocyanin

“…5b). One putative N-linkage site was also identified at position 166 in P. elephas α-type subunits 1, 2, and 3 (Meissner et al 2003), and β-type subunit a, b at position 470 in P. interuptus. Four potential N-glycosylation sites are present in each of the H. americanus pseudohemocyanin (Burmester 1999).…”