Human Mutation
Human Mutation
5 years ago

Exploring the limits of the usefulness of mutagenesis in studies of allosteric mechanisms

Exploring the limits of the usefulness of mutagenesis in studies of allosteric mechanisms
Aileen Y. Alontaga, Qingling Tang, Todd Holyoak, Aron W. Fenton
The outcome of structure-guided mutational analyses is often used in support of postulated mechanisms of protein allostery. However, the limits of how informative mutations can be in understanding allosteric mechanisms are not completely clear. Here, we report an exercise to evaluate whether mutational data can support a simplistic mechanistic model, developed with minimal data inputs. Due to the lack of a mechanism to explain how alanine allosterically modifies the affinity of human liver pyruvate kinase (approved symbol PKLR) for its substrate, phosphoenolpyruvate, we proposed a speculative allosteric mechanism for this system. Within the allosteric amino-acid-binding site (something in the effector site must, of necessity, contribute to the allosteric mechanism), we implemented multiple mutational strategies: (1) site-directed random mutagenesis at positions that contact bound alanine and (2) mutations to probe specific questions. Despite acknowledged inadequacies used to formulate the speculative mechanism, many mutations modified the allosteric coupling constant (Qax) consistent with that mechanism. The observed support for this speculative mechanism leaves us to ponder the best use of mutational data in structure–function studies of allosteric mechanisms. The mutational databank derived from this exercise has an independent value for training and testing algorithms specific to allostery. A simplistic allosteric mechanism model based on allosterically-relevant plasticity. This model was developed with acknowledged insufficient structural insights, but provided a framework for mutational probings of the allosteric binding site. Although we provide data that is not consistent with this model, the responses of many mutant proteins could be viewed as consistent, raising questions about how mutational probings should be used in studies of allosteric mechanisms.

Publisher URL: http://onlinelibrary.wiley.com/resolve/doi

DOI: 10.1002/humu.23239

You might also like
Discover & Discuss Important Research

Keeping up-to-date with research can feel impossible, with papers being published faster than you'll ever be able to read them. That's where Researcher comes in: we're simplifying discovery and making important discussions happen. With over 19,000 sources, including peer-reviewed journals, preprints, blogs, universities, podcasts and Live events across 10 research areas, you'll never miss what's important to you. It's like social media, but better. Oh, and we should mention - it's free.

  • Download from Google Play
  • Download from App Store
  • Download from AppInChina

Researcher displays publicly available abstracts and doesn’t host any full article content. If the content is open access, we will direct clicks from the abstracts to the publisher website and display the PDF copy on our platform. Clicks to view the full text will be directed to the publisher website, where only users with subscriptions or access through their institution are able to view the full article.