Structures of two elapid snake venom metalloproteases with distinct activities highlight the disulfide patterns in the D domain of ADAMalysin family proteins

Abstract

The structures of snake venom metalloproteases (SVMPs) are proposed to be useful models to understand the structural and functional relationship of ADAM (a disintegrin and metalloprotease) which are membrane-anchored proteins involved in multiple human diseases. We have purified, sequenced and determined the structures of two new P-III SVMPs - atragin and kaouthiagin-like (K-like) from Naja atra. Atragin exhibits a known C-shaped topology, whereas K-like adopts an I-shaped conformation because of the distinct disulfide pattern in the disintegrin-like (D) domain. K-like exhibits an enzymatic specificity toward pro-TNFalpha with less inhibition of cell migration, but atragin shows the opposite effect. The specificity of the enzymatic activity is indicated to be dominated mainly by the local structures of SVMP in the metalloprotease (M) domain, whereas the hyper-variable region (HVR) in the cysteine-rich (C) domain is involved in a cell-migration activity. We demonstrate also a pH-dependent enzymatic activity of atragin that we correlate with the structural dynamics of a Zn(2+)-binding motif and the Met-turn based on the structures determined with a pH-jump method. The structural variations between the C- and I-shapes highlight the disulfide bond patterns in the D domain of the ADAM/adamalysin/reprolysins family proteins.

Antibodies binding the ADAM10 substrate recognition domain inhibit Eph function

“…Interestingly, considerably higher affinity of these mAbs for the isolated C domain compared to the full-length extracellular domain, principally due to a higher association rate, suggests the binding epitope may be masked in much of the full-length protein, and thus dependent on the conformation of the ADAM10 ECD. Although the structure of the full ADAM10 ECD is yet to be solved, functional studies of the close relative ADAM17 suggest activity-dependent conformation changes (Wang et al, 2009;Willems et al, 2010), and structures of related snake venom metalloproteases also suggest distinct open and closed conformations that are suggested to regulate substrate access to the C domain (Guan et al, 2010), a notion which would be consistent with the binding behaviour of our mAbs to this region.…”

Cobra venom proteome and glycome determined from individual snakes of Naja atra reveal medically important dynamic range and systematic geographic variation

“…2 for the locations of venom source). The purified venom protein was isolated from the crude venom following the procedures described previously [11][12][13]. The venom standard and commercial bivalent horse antivenom for clinical usage (bivalent for N. atra and B. multicinctus; highly purified F(ab′) 2 molecule; lot: FN0101) is a gift from Taiwan CDC.…”

“…The quantitation of venom components was performed as described before [39] with slight modifications. Briefly, the relative abundance of each component was determined by the ratio of the corresponding peak area to total peak area at 210 nm after calibration using toxins purified for spectroscopic and structural analyses in this lab as described in our previous publications [11][12][13]. SDS-PAGE analysis was performed on the fractionated protein collected from reverse-phase HPLC samples with reduced volume by using SpeedVac.…”

“…We previously determined the three-dimensional structure of two elapid P-III type SVMP, e.g., K-like (PDB: 3K7N) and atragin (PDB: 3K7L), with distinct protease activities [12]. The electron densities of the two structures indicated the existence of two N-glycans at N319 and N490 for the K-like protein and one N-glycan for the atragin protein at N436 (see also Fig.…”

“…Many studies have been performed to shed light on the structure and function relationships of cobra venom toxins. The three-dimensional structures of most toxic components, including NTXs [9], PLA2s [10], CTXs (with different homologues of A1, A2, A3, A4, A5 and A6) [11], snake venom metalloproteinase (SVMP; K-like and atragin) [12], cysteine-rich secretory protein (CRISP) [13], and venom nerve growth factor (vNGF) [14] from Naja atra, are available. In addition, the genome from the king cobra (Ophiophagus hannah) [15], transcriptome from N. atra [16], and proteomes from African (Naja mossambica, Naja nigricollis, Naja katiensis, Naja nubiae, Naja pallida, and Naja haje) [17] and southeast Asian cobras (Naja kaouthia and Naja sumatrana) [7,18] have also been reported recently.…”