Low Resolution Crystal Structure of Arenicola Erythrocruorin: Influence of Coiled Coils on the Architecture of a Megadalton Respiratory Protein

Abstract

Annelid erythrocruorins are extracellular respiratory complexes assembled from 180 subunits into hexagonal bilayers. Cryo-electron microscopic experiments have identified two different architectural classes. In one, designated type I, the vertices of the two hexagonal layers are partially staggered, with one hexagonal layer rotated by about 16 degrees relative to the other layer, whereas in the other class, termed type II, the vertices are essentially eclipsed. We report here the first crystal structure of a type II erythrocruorin, that from Arenicola marina, at 6.2 A resolution. The structure reveals the presence of long continuous triple-stranded coiled-coil "spokes" projecting towards the molecular center from each one-twelfth unit; interdigitation of these spokes provides the only contacts between the two hexagonal layers of the complex. This arrangement contrasts with that of a type I erythrocruorin from Lumbricus terrestris in which the spokes are broken into two triple-stranded coiled coils with a disjointed connection. The disjointed connection allows formation of a more compact structure in the type I architecture, with the two hexagonal layers closer together and additional extensive contacts between the layers. Comparison of sequences of the coiled-coil regions of various linker subunits shows that the linker subunits from type II erythrocruorins possess continuous heptad repeats, whereas a sequence gap places these repeats out of register in the type I linker subunits, consistent with a disjointed coiled-coil arrangement.

Recent New Characterizations on the Giant Extracellular Hemoglobin of Glossoscolex paulistus and Some Other Giant Hemoglobins from Different Worms

“…It is worthy of mention, that another extracellular hemoglobin from a sea worm, HbAm, has also been investigated by Zal et al [22,23]. In this case the structure belongs to class 2, where the two hexagonal layers are exactly eclipsed one relative to the other, as demonstrated in recent crystallographic studies at low resolution [24]. Besides, the assembly of the protomer corresponding to 1/12 of the whole HbAm oligomer is different: a dodecamer formed by two monomeric units a 1 and a 2 together with a trimer (T) gives (a 1 ) 3 (a 2 ) 6 T associated to an average of 3.5 linkers resulting in a structure of [(a 1 ) 3 (a 2 ) 6 T]L 3.5 [23,25].…”

“…These extracellular hemoglobins are constituted by a large number of globin-like subunits containing heme groups with MM in the range 15-19 kDa. These globin subunits form monomers of 16 kDa (d) and disulphide bound hetero-trimers of 51-52 kDa (abc), linked by non-heme structures (24)(25)(26)(27)(28)(29)(30)(31)(32), named linkers (L) [12,13]. Recent partial characterization of MM of HbGp by matrix assisted laser www.intechopen.com desorption time-of-flight mass spectrometry (MALDI-TOF-MS) confirmed the similarity of its subunits to those of homologous proteins of this class, mentioned above, especially HbLt [15].…”

Ferric species of the giant extracellular hemoglobin of Glossoscolex paulistus as function of pH: An EPR study on the irreversibility of the heme transitions

“…This is due to their extraordinary supramolecular masses, approximately 3.6 MDa; high cooperativity; redox and oligomeric stabilities, etc. These interesting characteristics are associated to the existence of a central substructure that maintains the total protein assembly (Ohtsuki and Crewe, 1983), which is dependent on the type of erythrocruorin, in agreement with the classification of Royer et al (2007). These relevant properties provoked the interest of various research groups in their respective studies in order to improve the knowledge of the structure-function relationship of hemoproteins.…”

Metals content of Glossoscolex paulistus extracellular hemoglobin: Its peroxidase activity and the importance of these ions in the protein stability

“…Eleven different concentrations for guaiacol (4,8,10,12,16,20,24,28,33,38,43 μmol L −1 ) were used, and a fixed one for H 2 O 2 (10 mmol L − 1 ). Kinetic parameters as the Michaelis-Menten constant K M and maximum velocity V max values for guaiacol were determined using the Lineweaver-Burk plot for the two-substrate mechanism followed by peroxidase [33][34][35][36].…”

“…In type II, there is no rotation between the two layers and the structures are eclipsed. It has been observed for the HBL Hbs from Alvinella pompejana (ApHb) [19] and Arenicola marina (HbAm) [20].…”