Targeting Ras-, Rho-, and Rab-family GTPases via a conserved cryptic pocket.

The family of Ras-like GTPases consists of over 150 different members, regulated by an even larger number of guanine exchange factors (GEFs) and GTPase-activating proteins (GAPs) that comprise cellular switch networks that govern cell motility, growth, polarity, protein trafficking, and gene expression. Efforts to develop selective small molecule probes and drugs for these proteins have been hampered by the high affinity of guanosine triphosphate (GTP) and lack of allosteric regulatory sites. This paradigm was recently challenged by the discovery of a cryptic allosteric pocket in the switch II region of K-Ras. Here, we ask whether similar pockets are present in GTPases beyond K-Ras. We systematically surveyed members of the Ras, Rho, and Rab family of GTPases and found that many GTPases exhibit targetable switch II pockets. Notable differences in the composition and conservation of key residues offer potential for the development of optimized inhibitors for many members of this previously undruggable family.

Molecular basis for the recruitment of the Rab effector protein WDR44 by the GTPase Rab11.

The formation of complexes between Rab11 and its effectors regulates multiple aspects of membrane trafficking, including recycling and ciliogenesis. WD repeat-containing protein 44 (WDR44) is a structurally uncharacterized Rab11 effector that regulates ciliogenesis by competing with prociliogenesis factors for Rab11 binding. Here, we present a detailed biochemical and biophysical characterization of the WDR44-Rab11 complex and define specific residues mediating binding. Using AlphaFold2 modeling and hydrogen/deuterium exchange mass spectrometry, we generated a molecular model of the Rab11-WDR44 complex. The Rab11-binding domain of WDR44 interacts with switch I, switch II, and the interswitch region of Rab11. Extensive mutagenesis of evolutionarily conserved residues in WDR44 at the interface identified numerous complex-disrupting mutations. Using hydrogen/deuterium exchange mass spectrometry, we found that the dynamics of the WDR44-Rab11 interface are distinct from the Rab11 effector FIP3, with WDR44 forming a more extensive interface with the switch II helix of Rab11 compared with FIP3. The WDR44 interaction was specific to Rab11 over evolutionarily similar Rabs, with mutations defining the molecular basis of Rab11 specificity. Finally, WDR44 can be phosphorylated by Sgk3, with this leading to reorganization of the Rab11-binding surface on WDR44. Overall, our results provide molecular detail on how WDR44 interacts with Rab11 and how Rab11 can form distinct effector complexes that regulate membrane trafficking events.

The Impact of Time to Anterior Cruciate Ligament Reconstruction on Return to Duty Among Active Duty Military Personnel.

INTRODUCTION: After an anterior cruciate ligament (ACL) injury, service members often undergo ACL reconstruction (ACLR) to restore knee stability, which is critical for performing physically demanding and unconventional military-specific tasks. Despite advancements in surgical techniques and rehabilitation protocols, a large portion of service members will not fully return to duty (RTD) post-ACLR and will receive a permanent profile restriction (PP) or undergo a medical evaluation board (MEB). The timing of ACLR is a modifiable factor that can potentially impact RTD and remains underexplored in this population. This study aimed to assess the relationship between the timing of ACLR and its impact on RTD and meniscal procedures performed at index ACLR. METHODS: This retrospective observational study was conducted among active duty military personnel who underwent primary ACLR at Madigan Army Medical Center between October 1, 2016, and December 31, 2022. The primary outcome was the number of RTD, PP, or MEB outcomes. Secondary outcomes included the incidence and type of meniscal procedure performed at index ACLR. Kruskal-Wallis analyses were employed to assess the relationships between time to ACLR and RTD, as well as the incidence and type of meniscal procedure performed. After separating time to ACLR into four distinct time-based groups (0-3 months, 3-6 months, 6-12 months, and >12 months), a chi-squared test with post hoc analysis via Dunn's test with Bonferroni correction was conducted to identify a time interval from injury to ACLR that impacted RTD. RESULTS: Initial analysis to assess the relationship between time to ACLR and outcome (RTD, PP, or MEB) were significant (P = .02). Subsequent analyses performed between 4 distinct time-based groups (0-3 months, 3-6 months, 6-12 months, and >12 months) were also significant (P = .03). Pairwise comparisons revealed an 80% rate of RTD in the 0-3 month group compared to a 53% RTD rate in 3-6 month group (P = .006). However, comparisons between the 3-6 month and 6-12 month group (P = .68) and between the 6-12 month and greater than 12 month groups were not significant (P = .80).Additionally, time to ACLR significantly differed between service members who did not undergo any concurrent meniscal procedure to those who had a meniscal debridement (P = .002), and to those who underwent meniscal repair (P = .02). There was no significant difference in time to ACLR between those who underwent debridement versus repair (P = .22). Patients without any meniscal procedure had an average surgery time of 175 days, compared to 240 days for those undergoing meniscal repair and 295 days for those with meniscal debridement. CONCLUSION: The findings from this novel study suggest that ACLR within 3 months after injury can improve the likelihood of RTD without limitations. The timing of ACLR can also impact the incidence and type of meniscal procedures, as patients who did not undergo any concomitant meniscal procedures underwent ACLR within 6 months after injury. This study offers valuable insight into the importance of earlier ACLR among service members to improve RTD rates and decrease additional concomitant meniscal procedures.

Molecular basis for differential activation of p101 and p84 complexes of PI3Kγ by Ras and GPCRs.

Class IB phosphoinositide 3-kinase (PI3Kγ) is activated in immune cells and can form two distinct complexes (p110γ-p84 and p110γ-p101), which are differentially activated by G protein-coupled receptors (GPCRs) and Ras. Using a combination of X-ray crystallography, hydrogen deuterium exchange mass spectrometry (HDX-MS), electron microscopy, molecular modeling, single-molecule imaging, and activity assays, we identify molecular differences between p110γ-p84 and p110γ-p101 that explain their differential membrane recruitment and activation by Ras and GPCRs. The p110γ-p84 complex is dynamic compared with p110γ-p101. While p110γ-p101 is robustly recruited by Gßγ subunits, p110γ-p84 is weakly recruited to membranes by Gßγ subunits alone and requires recruitment by Ras to allow for Gßγ activation. We mapped two distinct Gßγ interfaces on p101 and the p110γ helical domain, with differences in the C-terminal domain of p84 and p101 conferring sensitivity of p110γ-p101 to Gßγ activation. Overall, our work provides key insight into the molecular basis for how PI3Kγ complexes are activated.

Leg injuries and wound repair among cosmetid harvestmen (Arachnida, Opiliones, Laniatores).

Previous studies of leg injuries in harvestmen have focused on the fitness consequences for individuals that use autospasy (voluntary detachment of the leg) as a secondary defense mechanism. Leg damage among non-autotomizing species of laniatorean harvestmen has not been investigated. Under laboratory conditions, we damaged femur IV of Cynorta marginalis and observed with scanning electron microscopy (SEM) the changes in these wounds over ten days. We also used SEM to examine leg damage from individuals of three species of cosmetid harvestmen that were collected in the field. On the basis of changes in the external surface of the hemolymph coagulum, we classified these wounds as fresh (coagulum forming), recent (coagulum with smooth surface), older (coagulum is scale-like with visible cell fragments), and fully healed (scale replaced by new cuticle growth on the terminal stump). Our observations indicate that wound healing in harvestmen occurs in a manner comparable to that of other chelicerates. Leg injuries exhibited interspecific variation with respect to the overall frequency of leg wounds and the specific legs that were most commonly damaged. In addition, we measured walking and climbing speeds of adult C. marginalis and found that individuals with fresh injuries (lab-induced) to femur IV walked at speeds significantly slower than uninjured adults or individuals collected from the field that had fully healed wounds to a single leg. J. Morphol. 278:73-88, 2017. ©© 2016 Wiley Periodicals,Inc.