HIV-1 matrix protein p17 variants (vp17s) detected in HIV-1-infected patients with non-Hodgkin's lymphoma (HIV-NHL) display, differently from the wild-type protein (refp17), B cell growth-promoting activity. Biophysical analysis revealed that vp17s are destabilized as compared to refp17, motivating us to explore structure-function relationships. We used: biophysical techniques (circular dichroism (CD), nuclear magnetic resonance (NMR) and thermal/GuHCL denaturation) to study protein conformation and stability; Surface plasmon resonance (SPR) to study interactions; Western blot to investigate signaling pathways; and Colony Formation and Soft Agar assays to study B cell proliferation and clonogenicity. By forcing the formation of a disulfide bridge between Cys residues at positions 57 and 87 we obtained a destabilized p17 capable of promoting B cell proliferation. This finding prompted us to dissect refp17 to identify the functional epitope. A synthetic peptide (F1) spanning from amino acid (aa) 2 to 21 was found to activate Akt and promote B cell proliferation and clonogenicity. Three positively charged aa (Arg15, Lys18 and Arg20) proved critical for sustaining the proliferative activity of both F1 and HIV-NHL-derived vp17s. Lack of any interaction of F1 with the known refp17 receptors suggests an alternate one involved in cell proliferation. The molecular reasons for the proliferative activity of vp17s, compared to refp17, relies on the exposure of a functional epitope capable of activating Akt. Our findings pave the way for identifying the receptor(s) responsible for B cell proliferation and offer new opportunities to identify novel treatment strategies in combating HIV-related NHL.

Novel therapeutics are needed to treat pathologies associated with the Clostridioides difficile binary toxin (CDT), particularly when C. difficile infection (CDI) occurs in the elderly or in hospitalized patients having illnesses, in addition to CDI, such as cancer. While therapies are available to block toxicities associated with the large clostridial toxins (TcdA and TcdB) in this nosocomial disease, nothing is available yet to treat toxicities arising from strains of CDI having the binary toxin. Like other binary toxins, the active CDTa catalytic subunit of CDT is delivered into host cells together with an oligomeric assembly of CDTb subunits via host cell receptor-mediated endocytosis. Once CDT arrives in the host cell’s cytoplasm, CDTa catalyzes the ADP-ribosylation of G-actin leading to degradation of the cytoskeleton and rapid cell death. Although a detailed molecular mechanism for CDT entry and host cell toxicity is not yet fully established, structural and functional resemblances to other binary toxins are described. Additionally, unique conformational assemblies of individual CDT components are highlighted herein to refine our mechanistic understanding of this deadly toxin as is needed to develop effective new therapeutic strategies for treating some of the most hypervirulent and lethal strains of CDT-containing strains of CDI.

S100B, a biomarker of malignant melanoma, interacts with the p53 protein and diminishes its tumor suppressor function, which makes this S100 family member a promising therapeutic target for treating malignant melanoma. However, it is a challenge to design inhibitors that are specific for S100B in melanoma versus other S100-family members that are important for normal cellular activities. For example, S100A1 is most similar in sequence and structure to S100B, and this S100 protein is important for normal skeletal and cardiac muscle function. Therefore, a combination of NMR and computer aided drug design (CADD) was used to initiate the design of specific S100B inhibitors. Fragment-based screening by NMR, also termed “SAR by NMR,” is a well-established method, and was used to examine spectral perturbations in 2D [¹H, ¹⁵N]-HSQC spectra of Ca²⁺-bound S100B and Ca²⁺-bound S100A1, side-by-side, and under identical conditions for comparison. Of the 1000 compounds screened, two were found to be specific for binding Ca²⁺-bound S100A1 and four were found to be specific for Ca²⁺-bound S100B, respectively. The NMR spectral perturbations observed in these six data sets were then used to model how each of these small molecule fragments showed specificity for one S100 versus the other using a CADD approach termed Site Identification by Ligand Competitive Saturation (SILCS). In summary, the combination of NMR and computational approaches provided insight into how S100A1 versus S100B bind small molecules specifically, which will enable improved drug design efforts to inhibit elevated S100B in melanoma. Such a fragment-based approach can be used generally to initiate the design of specific inhibitors for other highly homologous drug targets.

Clostridioides difficile infection (CDI) is one of the five most urgent bacterial threats in the United States. Furthermore, hypervirulent CDI strains express a third toxin termed the C. difficile binary toxin (CDT), and its molecular mechanism for entering host cells is not fully elucidated. Like other AB-type binary toxins, CDT enters host cells via endosomes. Here we show via surface plasmon resonance and electrochemical impedance spectroscopy that the cell-binding component of CDT, termed CDTb, binds and form pores in lipid bilayers in the absence of its enzymatic component, CDTa. This occurs upon lowering free Ca²⁺ ion concentration, and not by decreasing pH, as found for other binary toxins (i.e., anthrax). Cryogenic electron microscopy (CryoEM), X-ray crystallography, and nuclear magnetic resonance (NMR) studies show that dissociation of Ca²⁺ from a single site in receptor binding domain 1 (RBD1) of CDTb triggers conformational exchange in CDTb. These and structure/function studies of a Ca²⁺-binding double mutant targeting RBD1 (i.e., D623A/D734A) support a model in which dissociation of Ca²⁺ from RBD1 induces dynamic properties in CDTb that enable it to bind and form pores in lipid bilayers.