Conversion of the enzyme guanylate kinase into a mitotic-spindle orienting protein by a single mutation that inhibits GMP-induced closing

Abstract

New protein functions can require complex sequence changes, but the minimal path is not well understood. The guanylate kinase enzyme (GK enz ), which catalyzes phosphotransfer from ATP to GMP, evolved into the GK domain (GK dom ), a protein-binding domain found in membrane associate guanylate kinases that function in mitotic spindle orientation and cell adhesion. Using an induced polarity assay for GK dom function, we show that a single serine to proline mutation is sufficient to switch extant GK enz into a functional GK dom . The mutation blocks catalysis (GK enz function) but allows protein binding and spindle orientation (GK dom function). Furthermore, whereas the GK enz undergoes a large closing motion upon GMP binding, fluorescence quenching and NMR demonstrate that the S → P mutation inhibits GMP-induced GK movements. Disrupting GK closing with a mutation at a different position also leads to GK dom function, suggesting that blocking the GK enz closing motion is sufficient for functional conversion of GK enz to GK dom . Although subtle changes in protein function can require complex sequence paths, our work shows that entirely new functions can arise from single mutations that alter protein dynamics.protein interactions | neofunctionalization | protein engineering | molecular evolution P roteins perform remarkably diverse functions including catalysis, small molecule binding, and protein recognition. Molecular evolution seeks to understand how the diversity of known protein functions arose, whereas the goal of protein engineering is to create new ones (1). An evolutionary process for creating new protein functions is the divergent evolution of duplicated genes, in which gene duplication is followed by functional evolution [neofunctionalization; (2)]. However, loss of function (pseudogene formation or nonfunctionalization) competes with functional evolution and may be much more likely (3). The degree to which neofunctionalization can "win out" over pseudogene formation depends on the number of mutations required to attain the new function and the possible paths in sequence space to the new function. Thus, neofunctionalization is a competitive process that is dependent on the number of required mutations and how protein function is achieved. Insight into the mechanisms of neofunctionalization should improve our ability to develop previously undescribed protein functions.We examined neofunctionalization in the context of a dramatic functional change: creation of a mitotic spindle-orienting protein from a nucleotide kinase within the membrane associated guanylate kinase (MAGUK) family of proteins. MAGUKs are proteins that mediate intricate multicellular functions such as cell junction formation and mitotic spindle orientation and have been found only in metazoans and choanoflagellates (4, 5).

Genetically modified proteins: functional improvement and chimeragenesis

“…71,72,73,74,75 Three mutants (I387A, I387C and I387S) of aminopeptidase from Bacillus subtilis (BSAP) with a broad substrate specificity toward p-nitroanilides (pNAs) derivatives of Leu, Arg and Lys hydrolyzed Phe-pNA, which was undetectable in wild-type BSAP (Table 1). 74 In docking simulation, bestatin was docked into the predicted structure of BSAP as the substrate analog.…”

“…The S68P mutation converted the guanylate kinase enzyme (GKenz), which catalyzes phosphotransfer from ATP to GMP, into a protein-binding domain (GKdom) found in membrane associate guanylate kinases (MAGUK) that function in mitotic spindle orientation and cell adhesion (Table 1). 71 A dramatic functional change suggested that the MAGUK family proteins with a defining protein-binding feature have diverged from the enzymes GKenz, which is broadly distributed throughout evolution. With respect to the all known GKenz sequences, Ser is conserved at this position.…”

“…Although the Pro mutation abrogated catalytic activity in GKenz as revealed by crystalization, it retained the ability to bind ATP and altered the protein's conformational response to ligand binding. 71 The mutations introduced into neprilysin (NEP), a transmembrane zinc metallopeptidase that degrades a wide range of peptides, have fundamentally altered the cleavage site preference of the enzyme (Table 1). 75 Cleavage of amyloid beta 1-40 (Ab1-40) at Phe20-Ala21 by double mutant G399V/G714K has not been previously observed in NEP that was largely driven by a decrease in K M , which may result from the substrate being able to adopt an altered binding conformation at the active site.…”

Evolution of a Protein Interaction Domain Family by Tuning Conformational Flexibility

“…Placing this large-effect mutation – s36P (ancestral state, lowercase; derived state, capitalized) – into Anc-gk dup results in a complete loss of the ancestral nucleotide kinase function and gain of the derived Pins binding function (Figure 1A) 6 . In the context of the extant yeast GK enzyme, this mutation causes it to lose the ability to close in response to binding of its nucleotide substrate 16 , pointing to a role for flexibility in the transition between the two functions.…”

Solution NMR Structures of Pyrenophora tritici-repentis ToxB and Its Inactive Homolog Reveal Potential Determinants of Toxin Activity

“…dynamics and function but not the global structure of the protein (37). ToxB appears to belong to this class of proteins whose activity could also be modulated by changes in dynamics rather than structure.…”