Venom proteomes of South and North American opisthoglyphous (Colubridae and Dipsadidae) snake species: A preliminary approach to understanding their biological roles

NMRstructure of bitistatin – a missing piece in the evolutionary pathway of snake venom disintegrins

“…The presence of PIII SVMPs in Viperidae (vipers and pit vipers) [12,13], Elapidae (cobras, kraits, Abbreviations ADAM, a disintegrin and metalloprotease; SVMP, snake venom metalloproteinase. coral snakes) [14], Colubridae (colubroid snakes) [15] and Atractaspididae (mole vipers, stiletto snakes) [16] supports the view that an early recruitment event of an ADAM gene predated the radiation of the advanced snakes (Caenophidia) [17] around the Cretaceous-Paleocene boundary, approximately 60 million years ago [18]. On the other hand, PII-SVMPs occur only in Viperidae venoms and cluster with PIIISVMPs into a monophyletic cladogram [10].…”

“…As an alternative to the failure to obtain a homogeneous bitistatin purified from its natural source, 15 Nlabeled bitistatin (full-length isoform D1, residues 1-83, UniProtKB accession code P17497, with six extra amino acids in the N-terminus: Their resonances were indirectly assigned at a later stage, during the structural calculation process and when the ensemble of structures for bitistatin A already showed a reasonably good precision to avoid potential bias, via long-range NOE correlations with residues spatially close in the structures using the solution structure of flavoridin (1FVL) [38] bitistatin-B. The full assignment of the backbone nitrogen atoms was possible for species B (= major species in 15 N-rbitistatin sample), whereas 86% of the nitrogen atoms were assigned for bitistatin A using natural abundance 15 N-spectra.…”

“…The full assignment of the backbone nitrogen atoms was possible for species B (= major species in 15 N-rbitistatin sample), whereas 86% of the nitrogen atoms were assigned for bitistatin A using natural abundance 15 N-spectra. Eighty-eight percent and 82% assignment was achieved for the Ca carbons of bitistatins A and B, respectively, via 13 C-HSQC spectra recorded with the natural abundance samples.…”

“…Positive E. coli Bl21(DE3) clones, shown by PCR to contain the bitistatin-thioredoxin fusion constructs, were grown overnight at 37°C in 15 NH 4 Cl-M9 minimal medium (Tracer Tec, Madrid, Spain) containing 100 lgÁmL À1 of ampicillin, 33 lgÁmL À1 kanamycin and 12 lgÁmL À1 tetracycline, followed by a 1 : 10 (v/v) dilution in the same medium. For the induction of the expression of the recombinant fusion proteins, IPTG was added to a final concentration of 1 mM and the cell suspensions were incubated for another 7 h at 37°C.…”

“…Concomitant to the structural divergence of the disintegrin scaffold, a restricted panel of b 1 and b 3 integrin inhibitory motifs (Fig. 1B) has evolved, via positive Darwinian evolution [27], at the apex of a mobile 11-residue disulfide-closed loop protruding [14][15][16][17] A from the protein core. The vast majority of single-chain (long, medium-sized and short) disintegrins express the RGD sequence, which represents the basal motif and blocks the adhesive function of integrins a 8 b 1 , a 5 b 1 , a v b 1 , a v b 3 and a IIb b 3 .…”