Summary Viral glycoproteins mediate entry by pH-activated or receptor-engaged activation and exist in metastable pre-fusogenic states that may be stabilized by directed rational design. As recently reported, the conformationally fixed HIV-1 envelope glycoprotein (Env) trimers in the pre-fusion state (SOSIP) display molecular homogeneity and structural integrity at relatively high levels of resolution. However, the SOSIPs necessitate full Env precursor cleavage, which requires endogenous furin over-expression. Here, we developed an alternative strategy using flexible peptide covalent linkage of Env subdomains to produce soluble, homogeneous and cleavage-independent Env mimics, called native flexibly linked (NFL) trimers, as vaccine candidates. This simplified design avoids the need for furin co-expression and, in one case, antibody affinity purification to accelerate trimer scale-up for preclinical and clinical applications. We have successfully translated the NFL design to multiple HIV-1 subtypes, establishing the potential to become a general method of producing native-like, well-ordered Env trimers for HIV-1 or other viruses.

Sudan virus (genus ebolavirus) is lethal, yet no monoclonal antibody is known to neutralize it. Here we describe antibody 16F6 that neutralizes Sudan virus and present its structure bound to the trimeric viral glycoprotein. Unexpectedly, the 16F6 epitope overlaps that of KZ52, the only other antibody against the GP1,2 core to be visualized. Further, both antibodies against this key GP1–GP2-bridging epitope neutralize at a post-internalization step, likely fusion.

Thiamin pyrophosphate 1 (Figure 1A) is an essential cofactor in all living systems1. Its biosynthesis involves the separate syntheses of the pyrimidine 2 and thiazole 3 precursors, which are then coupled2. Two biosynthetic routes to the thiamin thiazole have been identified. In prokaryotes, five enzymes act on three substrates to produce the thiazole via a complex oxidative condensation reaction, the mechanistic details of which are now well established2–6. In contrast, only one gene-product is involved in thiazole biosynthesis in eukaryotes (THI4p in Saccharomyces cerevisiae)7. Identification of three adenylated metabolites (structures 5, 12 and 17 in Figure 1B), co-purifying with THI4p, provided three molecular snapshots of the reaction pathway catalyzed by this protein. In addition, two partially active mutants were identified (C204A and H200N), which catalyzed the conversion of NAD (nicotinamide adenine dinucleotide) 6 and glycine 9 to an advanced intermediate 128. A mechanism for thiazole formation, consistent with these observations, is outlined in Figure 1B.8–11 However, the source of the thiazole sulfur remained elusive, precluding us from deciphering the subsequent steps leading to the adenylated thiazole 5. Here we report the preparation of fully active recombinant wild type THI4p, the identification of an iron-dependent sulfide transfer reaction from the protein to a reaction intermediate and the demonstration that THI4p is a suicidal enzyme undergoing only a single turnover.

The structure of tryptophan 2,3-dioxygenase (TDO) from Ralstonia metallidurans was determined at 2.4 A. TDO catalyzes the irreversible oxidation of l-tryptophan to N-formyl kynurenine, which is the initial step in tryptophan catabolism. TDO is a heme-containing enzyme and is highly specific for its substrate l-tryptophan. The structure is a tetramer with a heme cofactor bound at each active site. The monomeric fold, as well as the heme binding site, is similar to that of the large domain of indoleamine 2,3-dioxygenase, an enzyme that catalyzes the same reaction except with a broader substrate tolerance. Modeling of the putative (S)-tryptophan hydroperoxide intermediate into the active site, as well as substrate analogue and mutagenesis studies, are consistent with a Criegee mechanism for the reaction.