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Protein functions have evolved in part via domain recombination events. Such events, for example, recombine structurally independent functional domains and shuff le targeting, regulatory, and͞or catalytic functions. Domain recombination, however, can generate new functions, as implied by the observation of catalytic sites at interfaces of distinct folding domains. If useful to an evolving organism, such initially rudimentary functions would likely acquire greater efficiency and diversity, whereas the initially distinct folding domains would likely develop into single functional domains. This represents the probable evolution of the S1 serine protease family, whose two homologous ␤-barrel subdomains assemble to form the binding sites and the catalytic machinery. Among S1 family members, the contact interface and catalytic residues are highly conserved whereas surrounding surfaces are highly variable. This observation suggests a new strategy to engineer viable proteins with novel properties, by swapping folding subdomains chosen from among protein family members. Such hybrid proteins would retain properties conserved throughout the family, including folding stability as single domain proteins, while providing new surfaces amenable to directed evolution or engineering of specific new properties. We show here that recombining the Nterminal subdomain from coagulation factor X with the C-terminal subdomain from trypsin creates a potent enzyme (fXYa) with novel properties, in particular a broad substrate specificity. As shown by the 2.15-Å crystal structure, plasticity at the hydrophobic subdomain interface maintains activity, while surface loops are displaced compared with the parent subdomains. fXYa thus represents a new serine proteinase lineage with hybrid fX, trypsin, and novel properties.Consideration of hierarchical features of protein architecture is particularly important in the design of novel enzymatic functions (1, 2). The simplest protein design problems involve the recruitment of existing evolved structural and functional elements (domains or modules) in straightforward recombination of associated properties, for example the combination of separate targeting and effector domains in immunotoxin design (3) or the shuffling of cofactor binding domains of coagulation factors (4). Many applications would benefit from the recombination of properties within a single functional domain; however, recombination of hierarchical units smaller than functional domains (referred to here as subdomains) generally risks loss of function or folding stability. A cautious attempt in this direction is the recombination of subdomains selected from homologous proteins that themselves have diversified through evolution but that retain relatively conserved catalytic machinery and subdomain interactions. The evolution of protein function via domain duplication and͞or recombination has generated numerous functional domains that comprise two such subdomains (5-9). This strategy allows one to swap specific subdomain linked func...

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