Indeed, TM helices of many cell surface receptors are not merely inert anchors but play essential roles in receptor assembly and signal transmission. membrane protein that fuses viral and host cell membranes to initiate viral infection . Sequential binding of gp120 to receptor (CD4) and coreceptor (e.g., CCR5 or CXCR4) triggers large conformational changes in both gp120 and gp41, leading to fusion and viral entry [2-4]. The mature and functional Env spikes, (gp120/gp41)3, are the sole antigens on the virion surface and thus the sole candidates for B-cell based vaccine development [5, 6]. The native prefusion conformation of HIV-1 Env is recognized by most broadly neutralizing antibodies (bnAbs) [7-9], and it is generally believed to have the potential for inducing such antibody responses. Thus, production of a recombinant form that closely resembles the conformation of Env spikes on the surface of virions is an important objective in immunogen design. Gp140, the uncleaved ectodomain of (gp160)3 with both the transmembrane segment (TM) and the cytoplasmic tail (CT) truncated, has often been considered as a likely surrogate for the native Env spike. Most HIV-1 gp140 preparations, however, are unstable and conformationally heterogeneous Acetazolamide . We have previously screened many HIV-1 primary isolates and identified two (clade A 92UG037.8 and clade C C97ZA012) that yield stable and homogeneous gp140 trimers when produced recombinantly in human cells [11, 12]. Other strategies to produce stable soluble Env trimers include the SOSIP modifications, which require an added disulfide bond between gp120 and gp41, an Ile-to-Pro mutation in gp41, and deletion of the membrane proximal external region (MPER) [9, 13]. While these approaches have yielded some useful Env trimer immunogens [8, 14, 15], they cannot explain why most soluble HIV-1 Env trimers are unstable. Moreover, we have shown recently that truncation of the CT in the context of the full-length Envs has an unexpectedly large impact on the antigenic structure of the ectodomain . These data suggest that the transmembrane domain (TMD), the only direct link between the CT and the ectodomain, may have a greater role in stabilizing the entire Env and in maintaining its native antigenic structure than previously recognized. As a type I membrane protein, HIV-1 Env has a single Acetazolamide TM segment with a GxxxG motif, implicated in oligomeric assembly of TM helices , and a conserved, positively charged residue (usually arginine). The TM is also more conserved than a typical transmembrane anchor, suggesting it may have functions other than just spanning a lipid bilayer. Indeed, TM helices of many cell surface receptors are not merely inert anchors but play essential roles in receptor assembly and signal transmission. Examples include EGFR, integrins and Fas receptor [17-20], for which Rabbit Polyclonal to S6K-alpha2 association of TM helices correlates the conformation of the extracellular domain with the conformations of the membrane proximal regions and the cytoplasmic signaling motifs. A similar mechanism could also underlie our observation that CT truncation affects the antigenic surface of the ectodomain of HIV-1 Env on the opposite side Acetazolamide of the membrane . Structure of the transmembrane domain of HIV-1 Env To understand the physical coupling (conformation and/or dynamics) between the CT and the ectodomain, we have determined a structure of the TMD of HIV-1 Env (residues 677-716), reconstituted in bicelles, by NMR Acetazolamide (; Fig. 1). The TMD is a well-structured trimer. It shows two unusual features not seen with other known oligomeric transmembrane helices (TMHs). The first is that there is an arginine (R696) near the middle of each of the TMHs, suggesting three unbalanced charges in the hydrophobic core of the membrane if the Arg remains protonated. In our NMR structure, the tips of the long sidechains of these arginines are facing lipids, and each of them is surrounded by three hydrophobic residues (L692 and L695 from the same TMH and I697 from the neighboring TMH). A precise physical basis for the stability of R696 in the highly hydrophobic environment remains unclear, but the underlying mechanism must tolerate a Lys residue, which is present in some viral isolates at this position. In our nuclear Overhauser enhancement (NOE) experiment for arginines, the R696 epsilon protons showed clear water NOE even with a short NOE mixing time of 60 ms, indicating bound water molecule(s) at this Arg position. While a high-resolution.