The structure, kinetics and interactions of the ß-carboxysomal ß-carbonic anhydrase, CcaA.

CcaA is a ß-carbonic anhydrase (CA) that is a component of the carboxysomes of a subset of ß-cyanobacteria. This protein, which has a characteristic C-terminal extension of unknown function, is recruited to the carboxysome via interactions with CcmM, which is itself a γ-CA homolog with enzymatic activity in many, but not all cyanobacteria. We have determined the structure of CcaA from Synechocystis sp. PCC 6803 at 1.45 Å. In contrast with the dimer-of-dimers organization of most bacterial ß-CAs, or the loose dimer-of-dimers-of-dimers organization found in the plant enzymes, CcaA shows a well-packed trimer-of-dimers organization. The proximal part of the characteristic C-terminal extension is ordered by binding at a site that passes through the two-fold symmetry axis shared with an adjacent dimer; as a result, only one of a pair of converging termini can be ordered at any given time. Docking in Rosetta failed to find well-packed solutions, indicating that formation of the CcaA/CcmM complex probably requires significant backbone movements in at least one of the binding partners. Surface plasmon resonance experiments showed that CcaA forms a complex with CcmM with sub-picomolar affinity, with contributions from residues in CcmM's αA helix and CcaA's C-terminal tail. Catalytic characterization showed CcaA to be among the least active ß-CAs characterized to date, with activity comparable with the γ-CA, CcmM, it either complements or replaces. Intriguingly, the C-terminal tail appears to partly inhibit activity, possibly indicating a role in minimizing the activity of unencapsulated enzyme.

Ubiquitin Engineering for Interrogating the Ubiquitin-Proteasome System and Novel Therapeutic Strategies.

Protein turnover, a highly regulated process governed by the ubiquitin-proteasome system (UPS), is essential for maintaining cellular homeostasis. Dysregulation of the UPS has been implicated in various diseases, including viral infections and cancer, making the proteins in the UPS attractive targets for therapeutic intervention. However, the functional and structural redundancies of UPS enzymes present challenges in identifying precise drug targets and achieving target selectivity. Consequently, only 26S proteasome inhibitors have successfully advanced to clinical use thus far. To overcome these obstacles, engineered peptides and proteins, particularly engineered ubiquitin, have emerged as promising alternatives. In this review, we examine the impact of engineered ubiquitin on UPS and non-UPS proteins, as well as on viral enzymes. Furthermore, we explore their potential to guide the development of small molecules targeting novel surfaces, thereby expanding the range of druggable targets.

A Panel of Engineered Ubiquitin Variants Targeting the Family of Domains Found in Ubiquitin Specific Proteases (DUSPs).

Domains found in ubiquitin specific proteases (DUSPs) occur in seven members of the ubiquitin specific protease (USP) family. DUSPs are defined by a distinct structural fold but their functions remain largely unknown, although studies with USP4 suggest that its DUSP enhances deubiquitination activity. We used phage-displayed libraries of ubiquitin variants (UbVs) to derive protein-based tools to target DUSP family members with high affinity and specificity. We designed a UbV library based on insights from the structure of a previously identified UbV bound to the DUSP of USP15. The new library yielded 33 unique UbVs that bound to DUSPs from five different USPs (USP4, USP11, USP15, USP20 and USP33). For each USP, we were able to identify at least one DUSP that bound with high affinity and absolute specificity relative to the other DUSPs. We showed that UbVs targeting the DUSPs of USP15, USP11 and USP20 inhibited the catalytic activity of the enzyme, despite the fact that the DUSP is located outside of the catalytic domain. These findings provide an alternative means of inhibiting USP activity by targeting DUSPs, and this mechanism could be potentially extended other DUSP-containing USPs.