causes listeriosis, a potentially fatal food-borne disease. The condition is especially harmful to pregnant women. outbreaks can originate from diverse foods, highlighting the need for novel strategies to improve food safety. The first step in invasion is internalization of the bacteria, which is mediated by the interaction of the internalin family of virulence factors with host cell receptors. A crucial interaction for invasion of the placenta, and thus a target for therapeutic intervention, is between internalin B (InlB) and the receptor c-Met. Single-domain antibodies (VH, also called nanobodies, or sdAbs) from camel heavy-chain antibodies are a novel solution for preventing infections. The VH R303, R330, and R326 all bind InlB with high affinity; however, the molecular mechanism behind their mode of action was unknown. We demonstrate that despite a high degree of sequence and structural diversity, the VH bind a single epitope on InlB. A combination of gentamicin protection assays and florescent microscopy establish that InlB-specific VH inhibit invasion of HeLa cells. A high-resolution X-ray structure of VH R303 in complex with InlB showed that the VH binds at the c-Met interaction site on InlB, thereby acting as a competitive inhibitor preventing bacterial invasion. These results point to the potential of VH as a novel class of therapeutics for the prevention of listeriosis.

The histone mark H3K27me3 and its reader/writer Polycomb repressive complex 2 (PRC2) mediate widespread transcriptional repression in stem and progenitor cells. Mechanisms that regulate this activity are critical for hematopoietic development but poorly understood. Here we show that the E3 ubiquitin ligase FBXO11 relieves PRC2-mediated repression during erythroid maturation by targeting its newly identified substrate BAHD1, an H3K27me3 reader that recruits transcriptional co-repressors. Erythroblasts lacking FBXO11 are developmentally delayed, with reduced expression of maturation-associated genes, most of which harbor bivalent histone marks (activating H3K4me3 and repressive H3K27me3), bind BAHD1, and fail to recruit the erythroid transcription factor GATA1. The BAHD1 complex interacts physically with PRC2 and depletion of either component restores FBXO11-deficient erythroid gene expression. Our studies identify BAHD1 as a novel effector of PRC2-mediated repression and reveal how a single E3 ubiquitin ligase eliminates PRC2 repression at developmentally poised bivalent genes during erythropoiesis.

A distinguishing feature of camel (Camelus dromedarius) VHH domains are noncanonical disulfide bonds between CDR1 and CDR3. The disulfide bond may provide an evolutionary advantage, as one of the cysteines in the bond is germline encoded. It has been hypothesized that this additional disulfide bond may play a role in binding affinity by reducing the entropic penalty associated with immobilization of a long CDR3 loop upon antigen binding. To examine the role of a noncanonical disulfide bond on antigen binding and the biophysical properties of a VHH domain, we have used the VHH R303, which binds the Listeria virulence factor InlB as a model. Using site directed mutagenesis, we produced a double mutant of R303 (C33A/C102A) to remove the extra disulfide bond of the VHH R303. Antigen binding was not affected by loss of the disulfide bond, however the mutant VHH displayed reduced thermal stability (Tm = 12°C lower than wild‐type), and a loss of the ability to fold reversibly due to heat induced aggregation. X‐ray structures of the mutant alone and in complex with InlB showed no major changes in the structure. B‐factor analysis of the structures suggested that the loss of the disulfide bond elicited no major change on the flexibility of the CDR loops, and revealed no evidence of loop immobilization upon antigen binding. These results suggest that the noncanonical disulfide bond found in camel VHH may have evolved to stabilize the biophysical properties of the domain, rather than playing a significant role in antigen binding.

Therapeutic antibodies constitute one of the fastest areas of growth in the field of biologic drugs. A molecular understanding of how antibodies interact with their target antigens is known as epitope mapping. The data provided by epitope mapping is extremely valuable in the process of antibody humanization, as well as in vaccine design. In many cases the epitope recognized by the antibody is a complex, discontinuous 3D conformational epitope. Mapping the interactions of an antibody to a conformational epitope is difficult by many standard approaches. X-ray crystallography is considered to be the gold standard of epitope mapping as it can provide a near atomic resolution model of the antibody-antigen interaction. An X-ray structure allows for inspection of specific antibody-antigen interactions, even in the case of complex conformational epitopes. The method described here can be adapted for structure determination and epitope mapping of any antibody fragment to a simple or complex antigen.

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