Propeptide-Mediated Allosteric Regulation of Xylanase Xyl-1: An Integrated Experimental and Computational Analysis.

Most GH11 family endo-ß-1,4-xylanases contain a propeptide region linked to the N-terminal region. The mechanistic basis of this region harboring key regulation information for enzyme function, however, remains poorly understood. We reported an investigation on the allosteric regulation mechanism of the propeptide based on biochemical characterization, molecular dynamics simulations, and evolutionary analysis. We discovered that the mutant of truncated propeptide shows a remarkably increased thermal stability (melting temperature increased by 11.5 °C) and catalytic efficiency (1.7-fold kcat/Km value of wild type). Molecular dynamics simulations reveal that long-range fluctuations in the propeptide lead to a conformational perturbation in the catalytic pocket and the thumb region. The probability of sampling the active conformation during the glycosylation step is reduced (i.e., catalytic efficiency). In-depth sequence analysis indicates that the propeptide has a strong plasticity and degeneration trend, and propeptide truncation experiments of the homologous enzyme XynB validated the feasibility of the truncation strategy. This work reveals the role of GH11 family propeptides in functional regulation and provides a straightforward and practical method to increase the robustness of GH11 family xylanases.

Similar protein segments shared between domains of different evolutionary lineages.

The emergence of novel proteins, beyond these that can be readily made by duplication and recombination of preexisting domains, is elusive. De novo emergence from random sequences is unlikely because the vast majority of random chains would not even fold, let alone function. An alternative explanation is that novel proteins emerge by duplication and fusion of pre-existing polypeptide segments. In this case, traces of such ancient events may remain within contemporary proteins in the form of reused segments. Together with the late Dan Tawfik, we detected such similar segments, far shorter than intact protein domains, which are found in different environments. The detection of these, "bridging themes," was based on a unique search strategy, where in addition to searching for similarity of shared fragments, so-called "themes," we also explicitly searched for cases in which the sequence segments before and after the theme are dissimilar (both in sequence and structure). Here, using a similar strategy, we further expanded the search and discovered almost 500 additional "bridging themes," linking domains that are often from ancient folds. The themes, of 20 residues or more (average 53), do not retain their structure despite sharing 37% sequence identity on average. Indeed, conformation flexibility may confer an evolutionary advantage, in that it fits in multiple environments. We elaborate on two interesting themes, shared between Rossmann/Trefoil-Plexin-like domains and a ß-propeller-like domain. FOR A BROAD AUDIENCE: A fundamental question in molecular evolution is how protein domains emerged. Similar segments shared between domains of seemingly distinct origins, may offer clues, as these may be remnants of the evolutionary process through which these domains emerged. However, finding such cases is difficult. Here, we expand the set of such cases which we curated previously, adding segments shared between domains that are considered ancient.