Structural basis for the distinct roles of non-conserved Pro116 and conserved Tyr124 of BCH domain of yeast p50RhoGAP.

p50RhoGAP is a key protein that interacts with and downregulates the small GTPase RhoA. p50RhoGAP is a multifunctional protein containing the BNIP-2 and Cdc42GAP Homology (BCH) domain that facilitates protein-protein interactions and lipid binding and the GAP domain that regulates active RhoA population. We recently solved the structure of the BCH domain from yeast p50RhoGAP (YBCH) and showed that it maintains the adjacent GAP domain in an auto-inhibited state through the ß5 strand. Our previous WT YBCH structure shows that a unique kink at position 116 thought to be made by a proline residue between alpha helices α6 and α7 is essential for the formation of intertwined dimer from asymmetric monomers. Here we sought to establish the role and impact of this Pro116. However, the kink persists in the structure of P116A mutant YBCH domain, suggesting that the scaffold is not dictated by the proline residue at this position. We further identified Tyr124 (or Tyr188 in HBCH) as a conserved residue in the crucial ß5 strand. Extending to the human ortholog, when substituted to acidic residues, Tyr188D or Tyr188E, we observed an increase in RhoA binding and self-dimerization, indicative of a loss of inhibition of the GAP domain by the BCH domain. These results point to distinct roles and impact of the non-conserved and conserved amino acid positions in regulating the structural and functional complexity of the BCH domain.

Structural Basis for the Enzymatic Activity of the HACE1 HECT-Type E3 Ligase Through N-Terminal Helix Dimerization.

HACE1 is an ankyrin repeat (AKR) containing HECT-type E3 ubiquitin ligase that interacts with and ubiquitinates multiple substrates. While HACE1 is a well-known tumor suppressor, its structure and mode of ubiquitination are not understood. The authors present the cryo-EM structures of human HACE1 along with in vitro functional studies that provide insights into how the enzymatic activity of HACE1 is regulated. HACE1 comprises of an N-terminal AKR domain, a middle (MID) domain, and a C-terminal HECT domain. Its unique G-shaped architecture interacts as a homodimer, with monomers arranged in an antiparallel manner. In this dimeric arrangement, HACE1 ubiquitination activity is hampered, as the N-terminal helix of one monomer restricts access to the C-terminal domain of the other. The in vitro ubiquitination assays, hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis, mutagenesis, and in silico modeling suggest that the HACE1 MID domain plays a crucial role along with the AKRs in RAC1 substrate recognition.

A novel allosteric site employs a conserved inhibition mechanism in human kidney-type glutaminase.

Glutaminase catalyses the metabolic process called glutaminolysis. Cancer cells harness glutaminolysis to increase energy reserves under stressful conditions for rapid proliferation. Glutaminases are upregulated in many tumours. In humans, the kidney-type glutaminase (KGA) isoform is highly expressed in the kidney, brain, intestine, foetal liver, lymphocytes and in many tumours. Glutaminase inhibition is shown to be effective in controlling cancers. Previously, we and others reported the inhibition mechanism of KGA using various inhibitors that target the active and allosteric sites of the enzyme. Here, we report the identification of a novel allosteric site in KGA using the compound DDP through its complex crystal structure combined with mutational and hydrogen-deuterium exchange mass spectrometry studies. This allosteric site is located at the dimer interface, situated ~ 31 Å away from the previously identified allosteric site and ~ 32 Å away from the active site. Remarkably, the mechanism of inhibition is conserved, irrespective of which allosteric pocket is targeted, causing the same conformational changes in the key loop near the active site (Glu312-Pro329) and subsequent enzyme inactivation. Contrary to the previously identified allosteric site, the identified new allosteric site is primarily hydrophilic. This site could be effectively targeted for the synthesis of specific and potent water-soluble inhibitors of glutaminase, which will lead to the development of anticancer drugs.

Sequence preference and scaffolding requirement for the inhibition of human neutrophil elastase by ecotin peptide.

Human neutrophil elastase (hNE) is an abundant serine protease that is a major constituent of lung elastolytic activity. However, when secreted in excess, if not properly attenuated by selective inhibitor proteins, it can have detrimental effects on host tissues, leading to chronic lung inflammation and non-small cell lung cancer. To improve upon the design of inhibitors against hNE for therapeutic applications, here, we report the crystal structure of hNE in complex with an ecotin (ET)-derived peptide inhibitor. We show that the peptide binds in the nonprime substrate binding site. Unexpectedly, compared with full-length (FL) ET, we find that our short linear peptides and circular amide backbone-linked peptides of ET are incapable of efficient hNE inhibition. Our structural insights point to a preferred amino acid sequence and the potential benefit of a scaffold for optimal binding and function of the peptide inhibitor, both of which are retained in the FL ET protein. These findings will aid in the development of effective peptide-based inhibitors against hNE for targeted therapy.

Structural basis for p50RhoGAP BCH domain-mediated regulation of Rho inactivation.

Spatiotemporal regulation of signaling cascades is crucial for various biological pathways, under the control of a range of scaffolding proteins. The BNIP-2 and Cdc42GAP Homology (BCH) domain is a highly conserved module that targets small GTPases and their regulators. Proteins bearing BCH domains are key for driving cell elongation, retraction, membrane protrusion, and other aspects of active morphogenesis during cell migration, myoblast differentiation, and neuritogenesis. We previously showed that the BCH domain of p50RhoGAP (ARHGAP1) sequesters RhoA from inactivation by its adjacent GAP domain; however, the underlying molecular mechanism for RhoA inactivation by p50RhoGAP remains unknown. Here, we report the crystal structure of the BCH domain of p50RhoGAP Schizosaccharomyces pombe and model the human p50RhoGAP BCH domain to understand its regulatory function using in vitro and cell line studies. We show that the BCH domain adopts an intertwined dimeric structure with asymmetric monomers and harbors a unique RhoA-binding loop and a lipid-binding pocket that anchors prenylated RhoA. Interestingly, the ß5-strand of the BCH domain is involved in an intermolecular ß-sheet, which is crucial for inhibition of the adjacent GAP domain. A destabilizing mutation in the ß5-strand triggers the release of the GAP domain from autoinhibition. This renders p50RhoGAP active, thereby leading to RhoA inactivation and increased self-association of p50RhoGAP molecules via their BCH domains. Our results offer key insight into the concerted spatiotemporal regulation of Rho activity by BCH domain-containing proteins.