Structural rearrangements of HIV-1 glycoprotein Env promote viral entry through membrane fusion. Env is a symmetric homotrimer with each protomer composed of surface subunit gp120 and transmembrane subunit gp41. Cellular CD4- and chemokine receptor-binding to gp120 coordinate conformational changes in gp41, first to an extended prehairpin intermediate (PHI) and, ultimately, into a fusogenic trimer-of-hairpins (TOH). HIV-1 fusion inhibitors target gp41 in the PHI and block TOH formation. To characterize structural transformations into and through the PHI, we employed asymmetric Env trimers containing both high and low affinity binding sites for individual fusion inhibitors. Asymmetry was achieved using engineered Env heterotrimers composed of protomers deficient in either CD4- or chemokine receptor-binding. Linking receptor engagement to inhibitor affinity allowed us to assess conformational changes of individual Env protomers in the context of a functioning trimer. We found that the transition into the PHI could occur symmetrically or asymmetrically depending on the stoichiometry of CD4 binding. Sequential engagement of multiple CD4s promoted progressive exposure of individual fusion inhibitor binding sites in a CD4-dependent fashion. By contrast, engagement of only a single CD4 molecule led to a delayed, but symmetric, exposure of the gp41 trimer. This complex coupling between Env-CD4 interaction and gp41 exposure explained the multiphasic fusion-inhibitor titration observed for a mutant Env homotrimer with a naturally asymmetric gp41. Our results suggest that the spatial and temporal exposure of gp41 can proceed in a nonconcerted, asymmetric manner depending on the number of CD4s that engage the Env trimer. The findings have important implications for the mechanism of viral membrane fusion and the development of vaccine candidates designed to elicit neutralizing antibodies targeting gp41 in the PHI. For HIV, cellular invasion requires merging viral and cellular membranes, an event achieved through the activity of the viral fusion protein Env. Env consists of three gp120 and three gp41 subunits symmetrically arranged on the viral surface. The gp120 subunits bind cellular receptors, which, in turn, coordinate gp41 conformational changes that promote membrane fusion. Understanding these structural rearrangements illuminates the mechanism of viral membrane fusion, and also spurs development of targeted inhibitors of viral entry and vaccine candidates that elicit antiviral immune responses. In this study, we employed a novel strategy to investigate individual subunits in the context of functioning Env complexes. The strategy links distinct gp120-receptor interactions to conformational changes that expose specific gp41 subunits. We found that, despite the initial symmetric arrangement of its subunits, Env conformational changes most often proceed quite asymmetrically, leading to exposure of only one-third of the gp41 trimer for much of the fusion event. This finding might explain why attempts to elicit potent anti-HIV antibodies to a fully exposed gp41 trimer have been largely unsuccessful. The study gives us a glimpse of the early structural transitions leading to Env-mediated membrane fusion and provides a framework for interrogating the fusion proteins of other membrane-encapsulated viruses.

"Allosteric" was first introduced to mean the other site (i.e., a site distinct from the active or orthosteric site), an adjective for "regulation" to imply a regulatory outcome resulting from ligand binding at another site. That original idea outlines a system with two ligand-binding events at two distinct locations on a macromolecule (originally a protein system), which defines a four-state energy cycle. An allosteric energy cycle provides a quantifiable allosteric coupling constant and focuses our attention on the unique properties of the four equilibrated protein complexes that constitute the energy cycle. Because many observed phenomena have been referenced as "allosteric regulation" in the literature, the goal of this work is to use literature examples to explore which systems are and are not consistent with the two-ligand thermodynamic energy cycle-based definition of allosteric regulation. We emphasize the need for consistent language so comparisons can be made among the ever-increasing number of allosteric systems. Building on the mutually exclusive natures of an energy cycle definition of allosteric regulation versus classic two-state models, we conclude our discussion by outlining how the often-proposed Rube-Goldberg-like mechanisms are likely inconsistent with an energy cycle definition of allosteric regulation.

Antibody binding to a plasma metalloprotease ADAMTS13 is necessary for the development of immune thrombotic thrombocytopenic purpura (iTTP). Inhibition of ADAMTS13-mediated VWF cleavage by such antibodies clearly plays a role in the pathophysiology of the disease, though the mechanisms by which they inhibit ADAMTS13 enzymatic function are not fully understood. At least some immunoglobulin G-type antibodies appear to affect the conformational accessibility of ADAMTS13 domains involved in both substrate recognition and inhibitory antibody binding. We utilized single chain fragments of the variable region (scFv's) previously identified via phage display from patients with iTTP to explore the mechanisms of action of inhibitory human monoclonal antibodies. Using recombinant full-length ADAMTS13, truncated ADAMTS13 variants, and native ADAMTS13 in normal human plasma, we found that, regardless of the conditions tested, all three inhibitory monoclonal antibodies affect enzyme turnover rate much more than substrate recognition of von Willebrand factor (VWF). Hydrogen-deuterium exchange plus mass spectrometry (HX-MS) experiments with each of these inhibitory antibodies demonstrated that residues in the active site of the catalytic domain of ADAMTS13 are differentially exposed to solvent in the presence and absence of monoclonal antibody binding. These results support the hypothesis that inhibition of ADAMTS13 in iTTP may not necessarily occur because the antibodies directly prevent VWF binding, but instead rely on allosteric effects that impair VWF cleavage, likely by affecting the conformation of the catalytic center in the protease domain of ADAMTS13. Our findings provide novel insight into the mechanism of autoantibody-mediated inhibition of ADAMTS13 and pathogenesis of iTTP.

Antibody-mediated inhibition of von Willebrand factor (VWF) cleavage by ADAMTS-13 results in immune thrombotic thrombocytopenic purpura (iTTP). However, the effects of multiple antibody binding to ADAMTS-13 are not fully understood. To determine how multiple antibodies affect ADAMTS13 activity under various conditions. Single-chain fragments of the variable region isolated via phage display from patients with iTTP, FRETS-VWF73, native ADAMTS-13 in normal human plasma, and hydrogen-deuterium exchange plus mass spectrometry were used. We found that 2 stimulatory antibodies affect ADAMTS-13 turnover rate more than its substrate recognition. Hydrogen-deuterium exchange plus mass spectrometry revealed that 1 of these 2 stimulatory antibodies bound to the CUB2 domain that presumably interacts with the spacer domain of ADAMTS-13. Spacer domain is targeted by most inhibitory antibodies in iTTP. Both inhibitory and stimulating antibodies could bind ADAMTS-13 simultaneously but when both were present the inhibitory activity predominates. The antibody-mediated stimulation was lost, but the inhibition persisted when a modified substrate with the amino acid residue leucine at position 1603 of VWF was replaced by an alanine (VWF73-L1603A), interfering with active site binding. These results support the hypothesis that the mechanism of action of both stimulatory and inhibitory anti-ADAMTS-13 antibodies in iTTP is through allosteric modification of the catalytic domain and that inhibition of ADAMTS-13 dominates when both are present. Our findings may provide a new avenue of exploration to develop targeted diagnostic and therapeutic approaches in the management of iTTP.

Background and Objective: Thrombotic thrombocytopenic purpura (TTP) is a rare but debilitating thrombotic microangiopathy that results from severe deficiency of the enzyme ADAMTS13. The disorder was first described in the early 20^th century, but the pathophysiology of the disease has only been elucidated in the past three decades. In this narrative review, we will summarize the milestone moments in the history of TTP research and discovery. Methods: We searched literature using PubMed from 1924 to 2023 using the following free text searches: “thrombotic thrombocytopenic purpura”, “Moschcowitz disease”, and “thrombotic microangiopathy”. We found 6,917 peer-reviewed articles and sorted through these for relevant literature pertinent to the review. A total of 46 articles were included for review and the remainder were excluded. Key Content and Findings: The history of TTP research was reviewed, with a sampling of major events in the evolution of the understanding of the pathophysiology and treatment of the disease discussed here. There remains much to be learned about the nature of the disease in order to develop more specific and less harmful treatments. Conclusions: An overview of the major discoveries that have led to our current understanding of TTP reveals the results of collaboration of multiple groups of physicians and scientists through the past century, with additional breakthroughs likely to occur in the future because of that same collaborative spirit.