Croquemort elicits activation of the immune deficiency pathway in ticks.

The immune deficiency (IMD) pathway directs host defense in arthropods upon bacterial infection. In Pancrustacea, peptidoglycan recognition proteins sense microbial moieties and initiate nuclear factor-κB-driven immune responses. Proteins that elicit the IMD pathway in non-insect arthropods remain elusive. Here, we show that an Ixodes scapularis homolog of croquemort (Crq), a CD36-like protein, promotes activation of the tick IMD pathway. Crq exhibits plasma membrane localization and binds the lipid agonist 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol. Crq regulates the IMD and jun N-terminal kinase signaling cascades and limits the acquisition of the Lyme disease spirochete B. burgdorferi. Additionally, nymphs silenced for crq display impaired feeding and delayed molting to adulthood due to a deficiency in ecdysteroid synthesis. Collectively, we establish a distinct mechanism for arthropod immunity outside of insects and crustaceans.

Binding and Functional Folding (BFF): A Physiological Framework for Studying Biomolecular Interactions and Allostery.

EF-hand Ca2+-binding proteins (CBPs), such as S100 proteins (S100s) and calmodulin (CaM), are signaling proteins that undergo conformational changes upon increasing intracellular Ca2+. Upon binding Ca2+, S100 proteins and CaM interact with protein targets and induce important biological responses. The Ca2+-binding affinity of CaM and most S100s in the absence of target is weak (CaKD > 1 µM). However, upon effector protein binding, the Ca2+ affinity of these proteins increases via heterotropic allostery (CaKD < 1 µM). Because of the high number and micromolar concentrations of EF-hand CBPs in a cell, at any given time, allostery is required physiologically, allowing for (i) proper Ca2+ homeostasis and (ii) strict maintenance of Ca2+-signaling within a narrow dynamic range of free Ca2+ ion concentrations, [Ca2+]free. In this review, mechanisms of allostery are coalesced into an empirical "binding and functional folding (BFF)" physiological framework. At the molecular level, folding (F), binding and folding (BF), and BFF events include all atoms in the biomolecular complex under study. The BFF framework is introduced with two straightforward BFF types for proteins (type 1, concerted; type 2, stepwise) and considers how homologous and nonhomologous amino acid residues of CBPs and their effector protein(s) evolved to provide allosteric tightening of Ca2+ and simultaneously determine how specific and relatively promiscuous CBP-target complexes form as both are needed for proper cellular function.

Physiologically Relevant Free Ca2+ Ion Concentrations Regulate STRA6-Calmodulin Complex Formation via the BP2 Region of STRA6.

The interaction of calmodulin (CaM) with the receptor for retinol uptake, STRA6, involves an α-helix termed BP2 that is located on the intracellular side of this homodimeric transporter (Chen et al., 2016 [1]). In the absence of Ca2+, NMR data showed that a peptide derived from BP2 bound to the C-terminal lobe (C-lobe) of Mg2+-bound CaM (MgCaM). Upon titration of Ca2+ into MgCaM-BP2, NMR chemical shift perturbations (CSPs) were observed for residues in the C-lobe, including those in the EF-hand Ca2+-binding domains, EF3 and EF4 (CaKD = 60 ± 7 nM). As higher concentrations of free Ca2+ were achieved, CSPs occurred for residues in the N-terminal lobe (N-lobe) including those in EF1 and EF2 (CaKD = 1000 ± 160 nM). Thermodynamic and kinetic Ca2+ binding studies showed that BP2 addition increased the Ca2+-binding affinity of CaM and slowed its Ca2+ dissociation rates (koff) in both the C- and N-lobe EF-hand domains, respectively. These data are consistent with BP2 binding to the C-lobe of CaM at low free Ca2+ concentrations (<100 nM) like those found at resting intracellular levels. As free Ca2+ levels approach 1000 nM, which is typical inside a cell upon an intracellular Ca2+-signaling event, BP2 is shown here to interact with both the N- and C-lobes of Ca2+-loaded CaM (CaCaM-BP2). Because this structural rearrangement observed for the CaCaM-BP2 complex occurs as intracellular free Ca2+ concentrations approach those typical of a Ca2+-signaling event (CaKD = 1000 ± 160 nM), this conformational change could be relevant to vitamin A transport by full-length CaCaM-STRA6.

Specificity of Molecular Fragments Binding to S100B versus S100A1 as Identified by NMR and Site Identification by Ligand Competitive Saturation (SILCS).

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.