Human fatty acid synthase (FAS) is uniquely expressed at high levels in many tumor types. Pharmacological inhibition of FAS therefore represents an important therapeutic opportunity. The drug Orlistat, which has been approved by the US Food and Drug Administration, inhibits FAS, induces tumor cell-specific apoptosis and inhibits the growth of prostate tumor xenografts. We determined the 2.3-A-resolution crystal structure of the thioesterase domain of FAS inhibited by Orlistat. Orlistat was captured in the active sites of two thioesterase molecules as a stable acyl-enzyme intermediate and as the hydrolyzed product. The details of these interactions reveal the molecular basis for inhibition and suggest a mechanism for acyl-chain length discrimination during the FAS catalytic cycle. Our findings provide a foundation for the development of new cancer drugs that target FAS.

Development of an HIV vaccine is a global priority. A major roadblock to a vaccine is an inability to induce protective broadly neutralizing antibodies (bnAbs). HIV gp41 bnAbs have characteristics that predispose them to be controlled by tolerance. We used gp41 2F5 bnAb germline knock-in mice and macaques vaccinated with immunogens reactive with germline precursors to activate neutralizing antibodies. In germline knock-in mice, bnAb precursors were deleted, with remaining anergic B cells capable of being activated by germline-binding immunogens to make gp41-reactive immunoglobulin M (IgM). Immunized macaques made B cell clonal lineages targeted to the 2F5 bnAb epitope, but 2F5-like antibodies were either deleted or did not attain sufficient affinity for gp41-lipid complexes to achieve the neutralization potency of 2F5. Structural analysis of members of a vaccine-induced antibody lineage revealed that heavy chain complementarity-determining region 3 (HCDR3) hydrophobicity was important for neutralization. Thus, gp41 bnAbs are controlled by immune tolerance, requiring vaccination strategies to transiently circumvent tolerance controls.

In trying to assess the structural integrity of electrospun type II collagen scaffolds, a modified but new technique for cross-linking collagen has been developed. Carbodiimides have been previously used to cross-link collagen in gels and in lyophilized native tissue specimens but had not been used for electrospun mats until recently. This cross-linking agent, and in particular 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), is of extreme interest, especially for tissue-engineered scaffolds composed specifically of native polymers (e.g., collagen), because it is a zero-length cross-linking agent that has not been shown to cause any cytotoxic reactions. The unique aspect of the cross-linking protocol in this study involves the use of ethanol as the solvent for the cross-linking agent, because the pure collagen electrospun mats immediately disintegrate when placed in an aqueous solution. This study examines 2 concentrations of EDC with and without the addition of N-hydroxysuccinimide to the reaction (which has been shown to result in higher cross-linking yields in aqueous solutions) to test the hypothesis that the use of EDC in a nonaqueous solution will cross-link electrospun type II collagen fibrous matrices in a comparable manner to typical glutaraldehyde fixation protocols. The use of EDC is compared with the cross-linking effects of glutaraldehyde via mechanical testing (uniaxial tensile testing) and biochemical testing (analysis of the percentage of free amino groups). The stress-strain curves of the cross-linked samples demonstrated uniaxial tensile behavior more characteristic of native tissue than do the dry, untreated samples. The heated, 50% glutaraldehyde cross-linking protocol resulted in a mean peak stress of 0.76 MPa, a mean strain at break of 127.30%, and a mean tangential modulus of 0.89 MPa; mean values for the samples treated with the EDC protocols ranged from 0.35 to 0.60 MPa for peak stress, from 111.83 to 159.23% for strain at break, and from 0.57 to 0.92 MPa for tangential modulus. Low and high concentrations (20 mM and 200 mM, respectively) of EDC alone were comparable in extent of cross-linking (29% and 29%, respectively) to the heated 50% glutaraldehyde cross-linking protocol (30% cross-linked).

HIV-1 mucosal transmission begins with virus or virus-infected cells moving through mucus across mucosal epithelium to infect CD4+ T cells. Although broadly neutralizing antibodies (bnAbs) are the type of HIV-1 antibodies that are most likely protective, they are not induced with current vaccine candidates. In contrast, antibodies that do not neutralize primary HIV-1 strains in the TZM-bl infection assay are readily induced by current vaccine candidates and have also been implicated as secondary correlates of decreased HIV-1 risk in the RV144 vaccine efficacy trial. Here, we have studied the capacity of anti-Env monoclonal antibodies (mAbs) against either the immunodominant region of gp41 (7B2 IgG1), the first constant region of gp120 (A32 IgG1), or the third variable loop (V3) of gp120 (CH22 IgG1) to modulate in vivo rectal mucosal transmission of a high-dose simian-human immunodeficiency virus (SHIV-BaL) in rhesus macaques. 7B2 IgG1 or A32 IgG1, each containing mutations to enhance Fc function, was administered passively to rhesus macaques but afforded no protection against productive clinical infection while the positive control antibody CH22 IgG1 prevented infection in 4 of 6 animals. Enumeration of transmitted/founder (T/F) viruses revealed that passive infusion of each of the three antibodies significantly reduced the number of T/F genomes. Thus, some antibodies that bind HIV-1 Env but fail to neutralize virus in traditional neutralization assays may limit the number of T/F viruses involved in transmission without leading to enhancement of viral infection. For one of these mAbs, gp41 mAb 7B2, we provide the first co-crystal structure in complex with a common cyclical loop motif demonstrated to be critical for infection by other retroviruses.

Summary Acyl-carrier-protein (ACP) represents one of the most highly conserved proteins across all domains of life and is nature's way of transporting hydrocarbon-chains in vivo. Notably, type II ACPs serve as a crucial interaction hub within primary cellular metabolism1 by communicating transiently between partner enzymes of the numerous biosynthetic pathways2,3. However, the highly transient nature of such interactions and the inherent conformational mobility of ACP2 have stymied previous attempts to structurally visualize ACP tied to an overall catalytic cycle. This is essential to understanding a fundamental aspect of cellular metabolism leading to compounds that are not only useful to the cell, but are also of therapeutic value. For example, ACP is central to the biosynthesis of the lipid A (endotoxin) component of lipopolysaccharides (LPS) in Gram-negative microorganisms, which is required for their growth and survival4,5 and is an activator of the mammalian host's immune system6,7, thus emerging as an important therapeutic target8-10. During lipid A synthesis (Raetz Pathway), ACP shuttles acyl-intermediates linked to its prosthetic 4′-phosphopantetheine group (4′-PPT)2 among four acyltransferases, including LpxD11. Here we report the crystal structures of three forms of Escherichia coli ACP engaging LpxD, which represent stalled substrate and liberated products along the reaction coordinate. The structures reveal the intricate interactions at the interface that optimally position ACP for acyl-delivery and that directly involve the pantetheinyl group. Conformational differences among the stalled ACPs provide the molecular basis for the association-dissociation process. An unanticipated conformational shift of 4′-phosphopantetheine groups within the LpxD catalytic chamber reveals an unprecedented role of ACP in product release.