Structure of lobster apocrustacyanin A₁using softer X-rays

Abstract

The molecular basis of the camouflage colouration of marine crustacea is often provided by carotenoproteins. The blue colour of the lobster carapace, for example, is intricately associated with a multimacromolecular 16‐mer complex of protein subunits each with a bound astaxanthin molecule. The protein subunits of crustacyanin fall into two distinct subfamilies, CRTC and CRTA. Here, the crystal structure solution of the A1 protein of the CRTC subfamily is reported. The problematic nature of the structure solution of the CRTC proteins (both C1 and A1) warranted consideration and the development of new approaches. Three putative disulfides per protein subunit were likely to exist based on molecular‐­homology modelling against known lipocalin protein structures. With two such subunits per crystallographic asymmetric unit, this direct approach was still difficult as it involved detecting a weak signal from these sulfurs and suggested the use of softer X‐rays, combined with high data multiplicity, as reported previously [Chayen et al. (2000), Acta Cryst. D56, 1064–1066]. This paper now describes the structure solution of CRTC in the form of the A1 dimer based on use of softer X‐­rays (2 Å wavelength). The structure solution involved a xenon derivative with an optimized xenon LI edge signal and a native data set. The hand of the xenon SIROAS phases was determined by using the sulfur anomalous signal from a high‐multiplicity native data set also recorded at 2 Å wavelength. For refinement, a high‐resolution data set was measured at short wavelength. All four data sets were collected at 100 K. The refined structure to 1.4 Å resolution based on 60 276 reflections has an R factor of 17.7% and an Rfree of 22.9% (3137 reflections). The structure is that of a typical lipocalin, being closely related to insecticyanin, to bilin‐binding protein and to retinol‐binding protein. This A1 monomer or dimer can now be used as a search motif in the structural studies of the oligomeric forms α‐ and β‐crustacyanins, which contain bound astaxanthin molecules.

The molecular basis of the coloration mechanism in lobster shell: β-crustacyanin at 3.2 Å resolution

“…The Ramachandran plot of the heterodimer (not shown) has 255 nonglycine residues in the most favored regions (80.3%), 60 residues in the additional allowed region (19.0%), 1 residue [Tyr-112 (A 1 )] in the generously allowed region (0.3%), and 1 residue [Tyr-110 (A 3 )] in the disallowed region (0.3%). This confirms that it is a feature of the lipocalin family to have these two residues as outliers (20). The secondary structure elements were assigned by PROCHECK (27).…”

“…Indeed, structure solution using the molecular replacement program AMORE (18), as implemented in the CCP4 package (19), verified the occurrence of only one dimer per asymmetric unit. The 1.4 Å refined model of apoCR A 1 (20) was used as search model in the polyalanine form. There were two clear solutions from which the unit cell packing was determined.…”

Vertebrate and invertebrate carotenoid-binding proteins

“…Crustacyanin has a molecular weight of about 320 kDa, and it is a multi-macromolecular 16-mer complex of protein subunits with 16 bound astaxanthin molecules. It was proposed that α-crustacyanin consists of two groups of homologous proteins (CRTC, containing apoproteins C 1 , C 2 and A 1 , and CRTA, containing apoproteins A 2 and A 3 ) [43]. Cianci et al 2002 further reported the structural basis of the bathochromic shift and binding behavior based on subunits A 1 and A 3 of curstacyanin protein and several different perturbations of the carotenoid molecules [44].…”

“…Vertebrates and invertebrates generally exhibit very limited capacity to metabolize carotenoids enzymatically, and relatively few carotenoid-specific metabolic enzymes have been reported [42][43]. Nonspecific esterases likely mediate xanthophyll ester cleavage in the gut, and some cells such as bird photoreceptors and frog retinal pigment epithelium re-esterify xanthophylls delivered to the tissue [17,45] Unlike mammals, birds possess a robust ability to add and remove oxygens on various carotenoids and to oxidize and reduce xanthophylls, presumably by enzymatically mediated pathways [46].…”

Selling candles in a post-Edison world: phasing with noble gases bound within engineered sites

“…The likelihood that a native protein will contain a noble-gas binding cavity has been estimated to be as high as 0.5 (Stowell et al, 1996). In practice, however, most structures that have been solved using noble-gas derivatives either bind gases (Vitali et al, 1991;Schiltz et al, 1994;Cohen et al, 2001) or apolar ligands (Bourguet et al, 1995;Weston et al, 1998;Cianci et al, 2001) as part of their biological function or contain fortuitous packing defects at subunit interfaces (Malashkevich et al, 1996;Wang et al, 2001;Mittl et al, 2002). Serine proteases comprise an interesting exception to these classes: in contrast to the apolar environments typically associated with binding sites, noble gases bind tightly within the highly polar catalytic sites of many of these enzymes (Schiltz et al, 1994).…”