A novel broad spectrum venom metalloproteinase autoinhibitor in the rattlesnake
 Crotalus atrox
 evolved via a shift in paralog function


 The complexity of snake venom composition reflects adaptation to the diversity of prey and may be driven at times by a coevolutionary arms race between snakes and venom-resistant prey. However, many snakes are also resistant to their own venom due to serum-borne inhibitors of venom toxins, which raises the question of how snake autoinhibitors maintain their efficacy as venom proteins evolve. To investigate this potential three-way arms race among venom, prey, and autoinhibitors, we have identified and traced the evolutionary origin of serum inhibitors of snake venom metalloproteinases (SVMPs) in the Western Diamondback rattlesnake
 Crotalus atrox
 which possesses the largest known battery of SVMP genes among crotalids examined. We found that
 C. atrox
 expresses five members of a Fetuin A-related metalloproteinase inhibitor family but that one family member, FETUA-3, is the major SVMP inhibitor that binds to approximately 20 different
 C. atrox
 SVMPs and inhibits activities of all three SVMP classes. We show that the
 fetua-3
 gene arose deep within crotalid evolution before the origin of New World species but, surprisingly,
 fetua-3
 belongs to a different paralog group than previously identified SVMP inhibitors in Asian and South American crotalids. Conversely, the
 C. atrox
 FETUA-2 ortholog of previously characterized crotalid SVMP inhibitors shows limited activity against
 C. atrox
 SVMPs. These results reveal that there has been a functional evolutionary shift in the major SVMP inhibitor in the
 C. atrox
 lineage as the SVMP family expanded and diversified in the
 Crotalus
 lineage. This broad-spectrum inhibitor may be of potential therapeutic interest.
