Harry B. Gristick

Cryo-EM structure of a CD4-bound open HIV-1 envelope trimer reveals structural rearrangements of the gp120 V1V2 loop.

Significance The HIV-1 envelope (Env) glycoprotein exists in multiple conformations on virion surfaces. Although the closed Env state is well characterized, less is known about open Env conformations stabilized by host receptor (CD4) binding. We solved an 8.9-Å structure of a partially open CD4-bound Env trimer by single particle cryo-EM. In the CD4-bound Env, the gp120 V1V2 loops were displaced by ∼40 Å from their positions at the trimer apex. The displaced V1V2 loops were at the sides of the open trimer in positions adjacent to, and interacting with, the three bound CD4s. These results are relevant to understanding CD4-induced conformational changes leading to coreceptor binding and fusion, and HIV-1 Env conformational dynamics, and describe a target structure relevant to drug design and vaccine efforts. The HIV-1 envelope (Env) glycoprotein, a trimer of gp120–gp41 heterodimers, relies on conformational flexibility to function in fusing the viral and host membranes. Fusion is achieved after gp120 binds to CD4, the HIV-1 receptor, and a coreceptor, capturing an open conformational state in which the fusion machinery on gp41 gains access to the target cell membrane. In the well-characterized closed Env conformation, the gp120 V1V2 loops interact at the apex of the Env trimer. Less is known about the structure of the open CD4-bound state, in which the V1V2 loops must rearrange and separate to allow access to the coreceptor binding site. We identified two anti–HIV-1 antibodies, the coreceptor mimicking antibody 17b and the gp120–gp41 interface-spanning antibody 8ANC195, that can be added as Fabs to a soluble native-like Env trimer to stabilize it in a CD4-bound conformation. Here, we present an 8.9-Å cryo-electron microscopy structure of a BG505 Env–sCD4–17b–8ANC195 complex, which reveals large structural rearrangements in gp120, but small changes in gp41, compared with closed Env structures. The gp120 protomers are rotated and separated in the CD4-bound structure, and the three V1V2 loops are displaced by ∼40 Å from their positions at the trimer apex in closed Env to the sides of the trimer in positions adjacent to, and interacting with, the three bound CD4s. These results are relevant to understanding CD4-induced conformational changes leading to coreceptor binding and fusion, and HIV-1 Env conformational dynamics, and describe a target structure relevant to drug design and vaccine efforts.

Coexistence of potent HIV-1 broadly neutralizing antibodies and antibody-sensitive viruses in a viremic controller.

Three new potent neutralizing antibodies neutralize autologous HIV-1 strains and contribute to viral control in an HIV-1 controller. Antibodies can hold HIV-1 at an impasse Neutralizing antibodies put selective pressure on pathogens to mutate and escape from immune detection, which is one of the reasons why HIV-1 infection is difficult to contain. In this issue, Freund et al. studied samples spanning almost a decade from an individual who naturally controls HIV-1 infection without progressing to AIDS. They discovered three potent antibodies coexisting with viral strains that were sensitive to antibody neutralization, indicating that these antibodies may be contributing to viral control. These antibodies were also able to prevent HIV-1 viremia in humanized mice, demonstrating that the antibodies may be beneficial as passive immunotherapy for infected individuals. Some HIV-1–infected patients develop broad and potent HIV-1 neutralizing antibodies (bNAbs) that when passively transferred to mice or macaques can treat or prevent infection. However, bNAbs typically fail to neutralize coexisting autologous viruses due to antibody-mediated selection against sensitive viral strains. We describe an HIV-1 controller expressing HLA-B57*01 and HLA-B27*05 who maintained low viral loads for 30 years after infection and developed broad and potent serologic activity against HIV-1. Neutralization was attributed to three different bNAbs targeting nonoverlapping sites on the HIV-1 envelope trimer (Env). One of the three, BG18, an antibody directed against the glycan-V3 portion of Env, is the most potent member of this class reported to date and, as revealed by crystallography and electron microscopy, recognizes HIV-1 Env in a manner that is distinct from other bNAbs in this class. Single-genome sequencing of HIV-1 from serum samples obtained over a period of 9 years showed a diverse group of circulating viruses, 88.5% (31 of 35) of which remained sensitive to at least one of the temporally coincident autologous bNAbs and the individual’s serum. Thus, bNAb-sensitive strains of HIV-1 coexist with potent neutralizing antibodies that target the virus and may contribute to control in this individual. When administered as a mix, the three bNAbs controlled viremia in HIV-1YU2–infected humanized mice. Our finding suggests that combinations of bNAbs may contribute to control of HIV-1 infection.

Crystal structure of ATP-bound Get3–Get4–Get5 complex reveals regulation of Get3 by Get4

Correct localization of membrane proteins is essential to all cells. Chaperone cascades coordinate the capture and handover of substrate proteins from the ribosomes to the target membranes, yet the mechanistic and structural details of these processes remain unclear. Here we investigate the conserved GET pathway, in which the Get4–Get5 complex mediates the handover of tail-anchor (TA) substrates from the cochaperone Sgt2 to the Get3 ATPase, the central targeting factor. We present a crystal structure of a yeast Get3–Get4–Get5 complex in an ATP-bound state and show how Get4 primes Get3 by promoting the optimal configuration for substrate capture. Structure-guided biochemical analyses demonstrate that Get4-mediated regulation of ATP hydrolysis by Get3 is essential to efficient TA-protein targeting. Analogous regulation of other chaperones or targeting factors could provide a general mechanism for ensuring effective substrate capture during protein biogenesis.

Differential gradients of interaction affinities drive efficient targeting and recycling in the GET pathway.

Significance Ensuring the accuracy of protein interaction cascades is a challenge in many cellular processes. This challenge is faced by the guided entry of tail-anchored (TA) protein (GET) pathway, in which the targeting factor Get3 must sequentially interact with three effector proteins to deliver an essential class of TA proteins to the membrane. Using fluorescence probes that quantitatively interrogate individual Get3–effector interactions, we show here that Get3 adopts discrete conformational states in response to substrate and nucleotide binding; these conformational states allow Get3 to generate differential gradients of interaction energies with distinct effectors, thus driving its cyclic and ordered interaction cascade. These results also explain why multiple effector proteins are needed for TA targeting and uncover a previously unidentified mechanism for recycling Get3 from the membrane. Efficient and accurate localization of membrane proteins requires a complex cascade of interactions between protein machineries. This requirement is exemplified in the guided entry of tail-anchored (TA) protein (GET) pathway, where the central targeting factor Get3 must sequentially interact with three distinct binding partners to ensure the delivery of TA proteins to the endoplasmic reticulum (ER) membrane. To understand the molecular principles that provide the vectorial driving force of these interactions, we developed quantitative fluorescence assays to monitor Get3–effector interactions at each stage of targeting. We show that nucleotide and substrate generate differential gradients of interaction energies that drive the ordered interaction of Get3 with successive effectors. These data also provide more molecular details on how the targeting complex is captured and disassembled by the ER receptor and reveal a previously unidentified role for Get4/5 in recycling Get3 from the ER membrane at the end of the targeting reaction. These results provide general insights into how complex protein interaction cascades are coupled to energy inputs in biological systems.

Asymmetric recognition of HIV-1 Envelope trimer by V1V2 loop-targeting antibodies

The HIV-1 envelope (Env) glycoprotein binds to host cell receptors to mediate membrane fusion. The prefusion Env trimer is stabilized by V1V2 loops that interact at the trimer apex. Broadly neutralizing antibodies (bNAbs) against V1V2 loops, exemplified by PG9, bind asymmetrically as a single Fab to the apex of the symmetric Env trimer using a protruding CDRH3 to penetrate the Env glycan shield. Here we characterized a distinct mode of V1V2 epitope recognition by the new bNAb BG1 in which two Fabs bind asymmetrically per Env trimer using a compact CDRH3. Comparisons between cryo-EM structures of Env trimer complexed with BG1 (6.2 Å resolution) and PG9 (11.5 Å resolution) revealed a new V1V2-targeting strategy by BG1. Analyses of the EM structures provided information relevant to vaccine design including molecular details for different modes of asymmetric recognition of Env trimer and a binding model for BG1 recognition of V1V2 involving glycan flexibility. DOI: http://dx.doi.org/10.7554/eLife.27389.001

Mechanism of Assembly of a Substrate Transfer Complex during Tail-anchored Protein Targeting

Background: Get4/5 is required for the efficient transfer of tail-anchored proteins to Get3. Results: The Get3·Get4/5 complex forms an intermediate mediated by electrostatic interactions. Conclusion: The rapid association of the Get3·Get4/5 intermediate complex is followed by a conformational change to the stable inhibited structure dominated by hydrophobic interactions. Significance: These results provide insight into the mechanism of tail-anchored protein targeting. Tail-anchored (TA) proteins, defined as having a single transmembrane helix at their C terminus, are post-translationally targeted to the endoplasmic reticulum membrane by the guided entry of TA proteins (GET) pathway. In yeast, the handover of TA substrates is mediated by the heterotetrameric Get4/Get5 complex (Get4/5), which tethers the co-chaperone Sgt2 to the targeting factor, the Get3 ATPase. Binding of Get4/5 to Get3 is critical for efficient TA targeting; however, questions remain about the formation of the Get3·Get4/5 complex. Here we report crystal structures of a Get3·Get4/5 complex from Saccharomyces cerevisiae at 2.8 and 6.0 Å that reveal a novel interface between Get3 and Get4 dominated by electrostatic interactions. Kinetic and mutational analyses strongly suggest that these structures represent an on-pathway intermediate that rapidly assembles and then rearranges to the final Get3·Get4/5 complex. Furthermore, we provide evidence that the Get3·Get4/5 complex is dominated by a single Get4/5 heterotetramer bound to one monomer of a Get3 dimer, uncovering an intriguing asymmetry in the Get4/5 heterotetramer upon Get3 binding. Ultrafast diffusion-limited electrostatically driven Get3·Get4/5 association enables Get4/5 to rapidly sample and capture Get3 at different stages of the GET pathway.

X-ray and EM structures of a natively glycosylated HIV-1 envelope trimer

The structural and biochemical characterization of broadly neutralizing anti-HIV-1 antibodies (bNAbs) has been essential in guiding the design of potential vaccines to prevent infection by HIV-1. While these studies have revealed critical mechanisms by which bNAbs recognize and/or accommodate N-glycans on the trimeric envelope glycoprotein (Env), they have been limited to the visualization of high-mannose glycan forms only, since heterogeneity introduced from the presence of complex glycans makes it difficult to obtain high-resolution structures. 3.5 and 3.9 Å resolution crystal structures of the HIV-1 Env trimer with fully processed and native glycosylation were solved, revealing a glycan shield of high-mannose and complex-type N-glycans that were used to define the complete epitopes of two bNAbs. Here, the refinement of the N-glycans in the crystal structures is discussed and comparisons are made with glycan densities in glycosylated Env structures derived by single-particle cryo-electron microscopy.

Structure of a Natively-glycosylated HIV-1 Env Reveals a New Mode for VH1-2 Antibody Recognition of the CD4 Binding Site Relevant to Vaccine Design

Background: Structural studies of broadly neutralizing antibodies (bNAbs) bound to Env trimers have revealed mechanisms by which bNAbs targeting various epitopes penetrate the glycan shield to either accommodate or include N-glycans in their epitopes. Although accessibility to the conserved host receptor (CD4) binding site (CD4bs) is restricted by surrounding glycans, VRC01-class bNAbs mimic CD4 binding to share a common mode of gp120 binding and glycan accommodation using a VH1-2*02- derived variable heavy (VH) domain. While attractive candidates for immunogen design, features of VRC01-class bNAbs such as a high degree of somatic hypermutation (SHM) and a short (5-residue) light chain (LC) complementarity determining region 3 (CDRL3) (found in only 1% of human LCs) suggest they might be difficult to elicit through vaccination. However, we recently isolated a VH1-2*02-derived CD4bs bNAb, named IOMA, that includes a normal-length (8 residues) CDRL3. Methods: We used X-ray crystallography to solve the first structure of a fully- and natively-glycosylated Env trimer in complex with IOMA, and the V3-loop-directed bNAb 10-1074. Results: Our structure revealed antibody-vulnerable glycan holes and roles of complex-type N-glycans on Env that are relevant to vaccine design, while also demonstrating that IOMA is a new class of CD4-mimetic bNAb that contains features of both VH1-2/VRC01-class and VH1-46/8ANC131-class bNAbs. Conclusions: Analysis of the native glycan shield on HIV-1 Env allows the first full description of the interplay between heterogeneous untrimmed high-mannose and complex-type N-glycans within the CD4bs, V3-loop, and other epitopes on Env. In addition, the structural characterization of IOMA revealed an alternative pathway from VRC01-class bNAbs relevant to vaccine design, which could more readily lead to an effective vaccine response due to higher frequencies of normal-length CDRL3s compared with the rare 5-residue CDRL3s required for VRC01-class bNAbs, and a lower need for SHMs.