Person:

Srouji, John R.

How do proteins evolve novel functions? To address this question, we are studying the evolution of a mammalian toxin, the serine protease BLTX [1], from the salivary glands of the North American shrew Blarina brevicauda. Here, we examine the molecular changes responsible for promoting BLTX toxicity. First, we show that regulatory loops surrounding the BLTX active site have evolved adaptively via acquisition of small insertions and subsequent accelerated sequence evolution. Second, these mutations introduce a novel chemical environment into the catalytic cleft of BLTX. Third, molecular-dynamic simulations show that the observed changes create a novel chemical and physical topology consistent with increased enzyme catalysis. Finally, we show that a toxic serine protease from the Mexican beaded lizard (GTX) [2] has evolved convergently through almost identical functional changes. Together, these results suggest that the evolution of toxicity might be predictable—arising via adaptive structural modification of analogous labile regulatory loops of an ancestral serine protease—and thus might aid in the identification of other toxic proteins.

Germ cells are the unique source of gametes for multicellular organisms and are specified through different mechanisms. One such specification mechanism is inheritance-mediated, in which the molecular factors necessary and sufficient to impart germ cell fate (collectively referred to as “germ plasm”) are maternally synthesized and deposited during oogenesis or early embryogenesis. Incorporation of this germ plasm into newly formed cells results in primordial germ cells. This mechanism stands in contrast to the predominant metazoan (and likely ancestral) mode of germ cell specification termed induction; germ cells specified via induction are the result of extracellular signaling from one embryonic tissue to another, triggering the expression of germ plasm components in the recipient cell type. In Drosophila melanogaster, the gene oskar (osk) is necessary and sufficient for organizing germ plasm in the oocyte, leading to primordial germ cell specification. While many investigators have offered thorough developmental and genetic investigations of osk, an understanding of the specific molecular mechanisms by which its protein products accumulate germ plasm is presently lacking. Oskar protein is uncharacterized, but is predicted to contain two well-folded domains: an N-terminal winged helix-turn-helix domain (the LOTUS/OST-HTH domain) and a C-terminal domain bearing sequence similarity to SGNH hydrolases (the lipase-related domain). The function of both domains is unknown, but previously published mutational data demonstrate that the lipase-related domain is crucial for Osk protein function. It is currently not known if the LOTUS/OST-HTH domain is necessary for the accumulation of germ plasm at the posterior pole. Using X-ray crystallography, I solved four structures of the LOTUS domain (corresponding to residues 139-241) and found that it forms a homodimer. In contrast to published characterized winged helix-turn-helix structures, the LOTUS homodimer conformation results from two β-hairpins (one from each protomer) forming a completed β-sheet. Analytical size exclusion chromatography, bacterial two-hybrid, and multi-angle light scattering experiments confirm that the LOTUS domain exists as a dimer in solution and combined with site-directed mutagenesis, the dimerization interface is indeed formed by a completed β-sheet as revealed by the crystal structures. Using a GAL4/UAS inducible D. melanogaster transgenic osk reporter, deletion of the LOTUS domain was found to abrogate the accumulation of germ plasm components, indicating that this domain is necessary for Oskar protein function. Furthermore, substituting the LOTUS domain with exogenous dimerization domains does not restore Osk activity. It is then postulated that oligomerization as mediated by the LOTUS domain constitutes at least one crucial aspect of its function within the context of full length Oskar protein.

ABSTRACT The Nudix homology clan encompasses over 80,000 protein domains from all three domains of life, defined by homology to each other. Proteins with a domain from this clan fall into four general functional classes: pyrophosphohydrolases, isopentenyl diphosphate isomerases (IDIs), adenine/guanine mismatch‐specific adenine glycosylases (A/G‐specific adenine glycosylases), and nonenzymatic activities such as protein/protein interaction and transcriptional regulation. The largest group, pyrophosphohydrolases, encompasses more than 100 distinct hydrolase specificities. To understand the evolution of this vast number of activities, we assembled and analyzed experimental and structural data for 205 Nudix proteins collected from the literature. We corrected erroneous functions or provided more appropriate descriptions for 53 annotations described in the Gene Ontology Annotation database in this family, and propose 275 new experimentally‐based annotations. We manually constructed a structure‐guided sequence alignment of 78 Nudix proteins. Using the structural alignment as a seed, we then made an alignment of 347 “select” Nudix homology domains, curated from structurally determined, functionally characterized, or phylogenetically important Nudix domains. Based on our review of Nudix pyrophosphohydrolase structures and specificities, we further analyzed a loop region downstream of the Nudix hydrolase motif previously shown to contact the substrate molecule and possess known functional motifs. This loop region provides a potential structural basis for the functional radiation and evolution of substrate specificity within the hydrolase family. Finally, phylogenetic analyses of the 347 select protein domains and of the complete Nudix homology clan revealed general monophyly with regard to function and a few instances of probable homoplasy. Proteins 2017; 85:775–811. © 2016 Wiley Periodicals, Inc.

ABSTRACT The nearly 50,000 known Nudix proteins have a diverse array of functions, of which the most extensively studied is the catalyzed hydrolysis of aberrant nucleotide triphosphates. The functions of 171 Nudix proteins have been characterized to some degree, although physiological relevance of the assayed activities has not always been conclusively demonstrated. We investigated substrate specificity for eight structurally characterized Nudix proteins, whose functions were unknown. These proteins were screened for hydrolase activity against a 74‐compound library of known Nudix enzyme substrates. We found substrates for four enzymes with k cat/K m values >10,000 M−1 s−1: Q92EH0_LISIN of Listeria innocua serovar 6a against ADP‐ribose, Q5LBB1_BACFN of Bacillus fragilis against 5‐Me‐CTP, and Q0TTC5_CLOP1 and Q0TS82_CLOP1 of Clostridium perfringens against 8‐oxo‐dATP and 3'‐dGTP, respectively. To ascertain whether these identified substrates were physiologically relevant, we surveyed all reported Nudix hydrolytic activities against NTPs. Twenty‐two Nudix enzymes are reported to have activity against canonical NTPs. With a single exception, we find that the reported k cat/K m values exhibited against these canonical substrates are well under 105 M−1 s−1. By contrast, several Nudix enzymes show much larger k cat/K m values (in the range of 105 to >107 M−1 s−1) against noncanonical NTPs. We therefore conclude that hydrolytic activities exhibited by these enzymes against canonical NTPs are not likely their physiological function, but rather the result of unavoidable collateral damage occasioned by the enzymes' inability to distinguish completely between similar substrate structures. Proteins 2016; 84:1810–1822. © 2016 The Authors Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.