Ligand-specific changes in conformational flexibility mediate long-range allostery in the lac repressor.

Biological regulation ubiquitously depends on protein allostery, but the regulatory mechanisms are incompletely understood, especially in proteins that undergo ligand-induced allostery with few structural changes. Here we used hydrogen-deuterium exchange with mass spectrometry (HDX/MS) to map allosteric effects in a paradigm ligand-responsive transcription factor, the lac repressor (LacI), in different functional states (apo, or bound to inducer, anti-inducer, and/or DNA). Although X-ray crystal structures of the LacI core domain in these states are nearly indistinguishable, HDX/MS experiments reveal widespread differences in flexibility.

Saturation mutagenesis of a predicted ancestral Syk-family kinase.

Many tyrosine kinases cannot be expressed readily in Escherichia coli, limiting facile production of these proteins for biochemical experiments. We used ancestral sequence reconstruction to generate a spleen tyrosine kinase (Syk) variant that can be expressed in bacteria and purified in soluble form, unlike the human members of this family (Syk and zeta-chain-associated protein kinase of 70 kDa [ZAP-70]). The catalytic activity, substrate specificity, and regulation by phosphorylation of this Syk variant are similar to the corresponding properties of human Syk and ZAP-70.

The SARS-CoV-2 spike reversibly samples an open-trimer conformation exposing novel epitopes.

Current COVID-19 vaccines and many clinical diagnostics are based on the structure and function of the SARS-CoV-2 spike ectodomain. Using hydrogen-deuterium exchange monitored by mass spectrometry, we have uncovered that, in addition to the prefusion structure determined by cryo-electron microscopy, this protein adopts an alternative conformation that interconverts slowly with the canonical prefusion structure. This new conformation-an open trimer-contains easily accessible receptor-binding domains.

Differences in the dynamics of the tandem-SH2 modules of the Syk and ZAP-70 tyrosine kinases.

The catalytic activity of Syk-family tyrosine kinases is regulated by a tandem Src homology 2 module (tSH2 module). In the autoinhibited state, this module adopts a conformation that stabilizes an inactive conformation of the kinase domain. The binding of the tSH2 module to phosphorylated immunoreceptor tyrosine-based activation motifs necessitates a conformational change, thereby relieving kinase inhibition and promoting activation.

Phosphorylation control of the ubiquitin ligase Cbl is conserved in choanoflagellates.

Cbl proteins are E3 ubiquitin ligases specialized for the regulation of tyrosine kinases by ubiquitylation. Human Cbl proteins are activated by tyrosine phosphorylation, thus setting up a feedback loop whereby the activation of tyrosine kinases triggers their own degradation. Cbl proteins are targeted to their substrates by a phosphotyrosine-binding SH2 domain.