Differences In Binding Specificity Research Articles

Differential Lipid Binding Specificities of RAP1A and RAP1B are Encoded by the Amino Acid Sequence of the Membrane Anchors.

RAP1 proteins belong to the RAS family of small GTPases that operate as molecular switches by cycling between GDP-bound inactive and GTP-bound active states. The C-terminal anchors of RAP1 proteins are known to direct membrane localization, but how these anchors organize RAP1 on the plasma membrane (PM) has not been investigated. Using high-resolution imaging, we show that RAP1A and RAP1B form spatially segregated nanoclusters on the inner leaflet of the PM, with further lateral segregation between GDP-bound and GTP-bound proteins. The C-terminal polybasic anchors of RAP1A and RAP1B differ in their amino acid sequences and exhibit different lipid binding specificities, which can be modified by single-point mutations in the respective polybasic domains (PBD). Molecular dynamics simulations reveal that single PBD mutations substantially reduce the interactions of the membrane anchors with the PM lipid phosphatidylserine. In summary, we show that aggregate lipid binding specificity encoded within the C-terminal anchor determines PM association and nanoclustering of RAP1A and RAP1B. Taken together with previous observations on RAC1 and KRAS, the study reveals that the PBD sequences of small GTPase membrane anchors can encode distinct lipid binding specificities that govern PM interactions.

Analysis of transferrin in urine in patients with bladder cancer using nanobodies

It is known that the saturation ratio of transferrin (Tf) with iron in human blood is an important clinical parameter. Specific antibodies can be used to analyze subtle changes in the relative abundance of different forms of transferrin potentially associated with a pathological process. Recently, the authors of this study were able to obtain and characterize highly specific single-domain antibodies (nanobodies) that predominantly recognize the iron-saturated (holo-Tf) or iron-unsaturated (apo-Tf) form of transferrin. In this work, under conditions closer to physiological than in previous experiments, we further demonstrated that these unique nanobodies have extremely high differential binding specificity for different forms of Tf in different human biological fluids. Using these nanobodies, we were able to analyze for the first time the relative abundance of transferrin forms in urine samples from patients with bladder cancer (BC). We have shown that an increase in the concentration of total Tf in urine samples normalized for creatinine is associated with the degree of progress and growth of malignancy of BC. In the samples of healthy donors and in the early stages of BC (G1), Tf is detected in much smaller amounts (compared to later stages) and only with additional concentration of the studied samples. For most of the studied urine samples from BC patients, it is expected (as previously shown in the case of Tf in the blood of terminal ovarian cancer patients) that the concentration of apo-Tf is clearly higher than holo-Tf, especially in the case of the most advanced muscle-invasive BC. It was a surprise for us that approximately equal amounts of apo-Tf and “holo-Tf” were found in the urine samples of some patients with BC. We hypothesized that the “holo-Tf” fraction in this case can be largely represented by “secondary complexes” formed by apo-Tf in combination with ions other than Fe3+, which accumulate in the urine of some cancer patients and are able to bind to apo-Tf, changing its conformation towards holo-Tf. Using inductively coupled plasma ionization mass spectroscopy (ICP-MS), we obtained the first results confirming our hypothesis. The “holo-Tf” preparation in these urine samples was found to be highly enriched in zinc and nickel. Also in this preparation, a relative enrichment in cadmium is observed, but it is present in much lower concentrations. The data obtained indicate that the nanobody used, recognizing predominantly the iron-saturated form of transferrin (holo-Tf), is also capable of binding transferrin in association with other metal ions that are different from iron. This ability could potentially open up new possibilities in studies of the relative abundance of various metal ions in association with transferrin in human biological fluids in normal and pathological conditions.

Analysis of Transferrin in the Urine of Patients with Bladder Cancer Using Nanobodies.

It is known that the saturation ratio of transferrin (Tf) with iron in human blood is an important clinical parameter. Specific antibodies can be used to analyze subtle changes in the relative abundance of different forms of transferrin potentially associated with a pathological process. Recently, the authors of this study were able to obtain and characterize highly specific single-domain antibodies (nanobodies) that predominantly recognize the iron-saturated (holo-Tf) or iron-unsaturated (apo-Tf) form of transferrin. In this work, under conditions closer to physiological than in the previous experiments, we further demonstrated that these unique nanobodies have extremely high differential binding specificity for different forms of Tf in different human biological fluids. Using these nanobodies, we were able to analyze for the first time relative abundance of the transferrin forms in urine samples from the patients with bladder cancer (BC). We have shown that increase in the concentration of total Tf in the urine samples normalized for creatinine is associated with the degree of progress and growth of malignancy of BC. In the samples of healthy donors and in the early stages of BC (G1), Tf is detected in much smaller amounts (compared to the later stages) and only with additional concentration of the studied samples. For most of the studied urine samples from the BC patients, it is expected (as previously shown in the case of Tf in the blood of terminal ovarian cancer patients) that the concentration of apo-Tf is clearly higher than holo-Tf, especially in the case of the most advanced muscle-invasive BC. It was a surprise for us that approximately equal amounts of apo-Tf and holo-Tf were found in the urine samples of some patients with BC. We hypothesized that the holo-Tf fraction in this case could be largely represented by the "secondary complexes" formed by apo-Tf in combination with ions other than Fe3+, which accumulate in the urine of some cancer patients and are able to bind to apo-Tf, changing its conformation towards holo-Tf. By using inductively coupled plasma mass spectroscopy (ICP-MS), we obtained first results confirming our hypothesis. Preparation of the holo-Tf in these urine samples was found to be highly enriched in zinc and nickel. Also, relative enrichment in cadmium has been observed in this preparation, but at much lower concentrations. The obtained data indicate that the used nanobody, while recognizing predominantly the iron-saturated form of transferrin (holo-Tf), is also capable of binding transferrin in association with other metal ions that are different from iron. This ability could potentially open up new possibilities for investigation of relative abundance of various metal ions in association with transferrin in human biological fluids in normal and pathological conditions.

Functional Analysis of Two Divergent C4 Isotypes in the Classical and Lectin Pathways of Complement Activation in the Common Carp (Cyprinus carpio)

In the evolution of the complement system, a major humoral innate immune factor, the existence of multiple isotypes of the complement components is considered as a key strategy to enhance innate immune defense. Complement C4 is also diversified in a wide range of vertebrate species including teleost fish, possibly supporting the robust activation mechanism of the complement. To better understand the functional diversity of C4 isotypes in the teleost complement system, two C4 isotypes, C4-1 and C4-2, sharing only 32% amino acid sequence identity, were examined for binding specificities towards model target molecules representing microbe antigens and towards Gram-positive and -negative bacteria. The results suggest that C4-1 and C4-2 behave similarly in binding to the tested targets, despite the predicted difference in binding specificity based on the thioester catalytic site. The participation of C4-1 in the classical and lectin pathways of complement activation was also explored using pathway-specific activating enzyme complexes, C1r/s and MBL-MASP2. As a result, C4-1 can be activated in both the classical and the lectin pathways, at higher efficiency in the classical pathway. Taken together, the present results imply that both C4-1 and C4-2 isotypes are fully functional in the complement activation cascades, probably playing comparable roles in innate immunity.

Next generation drug connectivity mapping for acquiring therapeutic agents to differentially regulate myelination.

Next generation drug connectivity mapping for acquiring therapeutic agents to differentially regulate myelination.

Molecular View into Preferential Binding of the Factor VII Gla Domain to Phosphatidic Acid.

Factor VII (FVII) is a serine protease with a key role in initiating the coagulation cascade. It is part of a family of vitamin K-dependent clotting proteins, which require vitamin K for formation of their specialized membrane-binding domains (Gla domains). Membrane binding of the FVII Gla domain is critical to the activity of FVII, mediating the formation of its complex with other clotting factors. While Gla domains among coagulation factors are highly conserved in terms of amino acid sequence and structure, they demonstrate differential binding specificity toward anionic lipids. Although most Gla domain-containing clotting proteins display a strong preference for phosphatidylserine (PS), it has been demonstrated that FVII and protein C instead bind preferentially to phosphatidic acid (PA). We have developed the first model of the FVII Gla domain bound to PA lipids in membranes containing PA, the highly mobile membrane mimetic model, which accelerates slow diffusion of lipids in molecular dynamics simulations and therefore facilitates the membrane binding process and enhances sampling of lipid interactions. Simulations were performed using atomic level molecular dynamics, requiring a fixed charge to all atoms. The overall charge assigned to each PA lipid for this study was -1. We also developed an additional model of the FVII Gla domain bound to a 1:1 PS/PC membrane and compared the modes of binding of PS and PA lipids to FVII, allowing us to identify potential PA-specific binding sites.

Dishevelled localization and function are differentially regulated by structurally distinct sterols

Aberrant cholesterol synthesis, characterized by depletion of cellular cholesterol and accumulation of sterol precursors, is directly linked to a group of rare human diseases exhibiting broad clinical phenotypes and developmental malformations. Despite decreased cholesterol content being shared across these diseases, patients exhibit distinct clinical manifestations associated with specific tissues or systems with varying degrees of phenotypic overlap. This tissue specificity may result from disease‐specific disruption of important critical signaling pathways due to the inability of sterol precursors to structurally compensate for cholesterol. We previously determined that the cholesterol precursor 7‐dehydrocholesterol (7DHC), which accumulates in Smith‐Lemli‐Opitz syndrome, perturbs Dishevelled 2 (DVL2) protein function due to reduced binding affinity of 7DHC to DVL2’s PDZ domain (Postsynaptic density 95, Disc large 1, and Zonular occludens 1 domain) in comparison to cholesterol, resulting in loss of canonical Wnt/β‐catenin signaling (1). We hypothesized that structural differences between cholesterol and alternative sterols will inhibit binding with DVL2 and other PDZ‐domain containing proteins, producing dire cellular consequences and directly affecting Wnt signaling (2). Protein homology modeling and ligand‐protein docking analysis suggested a differential binding affinity and specificity of DVL2 for cholesterol versus alternative sterols. Using PM‐mimetic vesicles incorporating cholesterol versus alternative sterols in liposome/DVL2 pulldowns, we experimentally observed a reduced binding affinity between DVL2 and alternate sterols. Surface plasmon resonance analysis to define the kinetics of protein‐sterol interaction corroborates in silicomodeling and pulldowns. Our data suggests orientation of the beta‐ and alpha‐sterol face within the DVL2‐PDZ domain binding pocket is predictive of DVL2‐sterol interaction. At the cellular level, we have also observed disruption of DVL2‐sterol binding shifts the subcellular localization of DVL2 from the plasma membrane to the nucleus. Increased nuclear DVL2 promotes non‐canonical WNT signaling and DVL2 protein‐protein binding with nuclear factors. Our work suggests that disrupted sterol homeostasis broadly impacts cellular signaling in a sterol‐specific and pathway‐specific manner. Defining the structural and biochemical requirements for sterol interactions with DVL2 and other cholesterol‐binding proteins will help define the critical role sterol homeostasis plays in human health.(1)Francis KR et al, Nature medicine. 2016;22(4):388‐96.(2) Sheng R et al, Nature communications. 2012;3:1249.

Kingella kingae PilC1 and PilC2 are adhesive multifunctional proteins that promote bacterial adherence, twitching motility, DNA transformation, and pilus biogenesis.

The gram-negative bacterium Kingella kingae is a leading cause of osteoarticular infections in young children and initiates infection by colonizing the oropharynx. Adherence to respiratory epithelial cells represents an initial step in the process of K. kingae colonization and is mediated in part by type IV pili. In previous work, we observed that elimination of the K. kingae PilC1 and PilC2 pilus-associated proteins resulted in non-piliated organisms that were non-adherent, suggesting that PilC1 and PilC2 have a role in pilus biogenesis. To further define the functions of PilC1 and PilC2, in this study we eliminated the PilT retraction ATPase in the ΔpilC1ΔpilC2 mutant, thereby blocking pilus retraction and restoring piliation. The resulting strain was non-adherent in assays with cultured epithelial cells, supporting the possibility that PilC1 and PilC2 have adhesive activity. Consistent with this conclusion, purified PilC1 and PilC2 were capable of saturable binding to epithelial cells. Additional analysis revealed that PilC1 but not PilC2 also mediated adherence to selected extracellular matrix proteins, underscoring the differential binding specificity of these adhesins. Examination of deletion constructs and purified PilC1 and PilC2 fragments localized adhesive activity to the N-terminal region of both PilC1 and PilC2. The deletion constructs also localized the twitching motility property to the N-terminal region of these proteins. In contrast, the deletion constructs established that the pilus biogenesis function of PilC1 and PilC2 resides in the C-terminal region of these proteins. Taken together, these results provide definitive evidence that PilC1 and PilC2 are adhesins and localize adhesive activity and twitching motility to the N-terminal domain and biogenesis to the C-terminal domain.

Molecular features underlying differential SHP1/SHP2 binding of immune checkpoint receptors.

A large number of inhibitory receptors recruit SHP1 and/or SHP2, tandem-SH2-containing phosphatases through phosphotyrosine-based motifs immunoreceptor tyrosine-based inhibitory motif (ITIM) and immunoreceptor tyrosine-based switch motif (ITSM). Despite the similarity, these receptors exhibit differential effector binding specificities, as exemplified by the immune checkpoint receptors PD-1 and BTLA, which preferentially recruit SHP2 and SHP1, respectively. The molecular basis by which structurally similar receptors discriminate SHP1 and SHP2 is unclear. Here, we provide evidence that human PD-1 and BTLA optimally bind to SHP1 and SHP2 via a bivalent, parallel mode that involves both SH2 domains of SHP1 or SHP2. PD-1 mainly uses its ITSM to prefer SHP2 over SHP1 via their C-terminal SH2 domains (cSH2): swapping SHP1-cSH2 with SHP2-cSH2 enabled PD-1:SHP1 association in T cells. In contrast, BTLA primarily utilizes its ITIM to prefer SHP1 over SHP2 via their N-terminal SH2 domains (nSH2). The ITIM of PD-1, however, appeared to be de-emphasized due to a glycine at pY+1 position. Substitution of this glycine with alanine, a residue conserved in BTLA and several SHP1-recruiting receptors, was sufficient to induce PD-1:SHP1 interaction in T cells. Finally, structural simulation and mutagenesis screening showed that SHP1 recruitment activity exhibits a bell-shaped dependence on the molecular volume of the pY+1 residue of ITIM. Collectively, we provide a molecular interpretation of the SHP1/SHP2-binding specificities of PD-1 and BTLA, with implications for the mechanisms of a large family of therapeutically relevant receptors.

The Distribution Profile of Glycoconjugates in the Testis of Brown-Banded Bamboo Shark (Chiloscyllium punctatum) by Using Lectin Histochemistry.

The testis of bamboo shark is characterized by diametric development leading to zonation architecture. Here, we investigated the staining pattern of 12 lectins in 6 groups of differential binding specificities within the germ, somatic, and interstitial cells of each zone. The neutral mucopolysaccharides appeared in the interstitial tissue in all the zones and became more significant in the spermatozoal–Sertoli cell junction. The cellular localization of the lectins varies in testicular zones and cell types. There was a gradual increase in glycosylation toward the degenerative zone. The increased intensity of most lectins in the interstitial cells indicates the association of glycoconjugates in their androgen-secreting activity. Statistical analyses showed a significant correlation between the groups of lectins and each lectin used, stronger response to lectins in the interstitial cells (ICs) than other cell types. Moreover, the response to glucosamine (GlcNAc), galactosamine (GalNAc), and fucose tended to be higher than glucose and galactose. Furthermore, the intensity of response was increased toward the degenerative zone. In addition, we can use peanut agglutinin (PNA) as an acrosomal marker in combination with other marker proteins for studying shark spermatogenesis. These findings refer to the crucial role of glycoconjugates in spermatogenesis in the bamboo shark testis.

IgG1 antibodies against phosphorylcholine are associated with protection in SLE and atherosclerosis: potential underlying mechanisms

Abstract Background The risk of cardiovascular disease (CVD) and atherosclerosis is very high in SLE. This is a clinical problem, and could also shed light on immunity and atherosclerosis in general. IgM antibodies against phosphorylcholine (anti-PC) may be protective in atherosclerosis, cardiovascular disease (CVD) and systemic lupus erythematosus (SLE). We here study IgG1 and IgG2 anti-PC, with focus on atherosclerosis and SLE. Methods We determined anti-PC by ELISA in 116 SLE-patients and 110 age- and sex-matched controls. For functional studies, we used three in-house generated, fully human monoclonal IgG1 anti-PC (A01, D05, E01). Apoptosis was induced in Jurkat T-cells and pre-incubated with A01, D05, E01 or isotype control IgG1 and effects on efferocytosis by human macrophages studied. Anti-PC peptide/protein characterization was determined using a proteomics de novo sequencing approach. Results IgG1 but not IgG2 anti-PC levels were higher among SLE patients (p=0.02). IgG1 anti-PC was negatively associated with SLICC and SLEDAI (OR: 2,978 CI: 0.876–10.098, OR: 5.108 CI 1.3 20.067 respectively) and negatively associated with CVD, atherosclerotic plaques and echolucent (potentially vulnerable plaques) but the association for the two former was not significant after controlling for confounders. D05 had maximum effect on macrophage efferocytosis efficiency, followed by A01 and E01. The monoclonal antibodies showed differential binding specificity to PC and PC associated neo-epitopes. Peptide analysis showed difference in the CDR3 region of the three anti-PC IgG1 clones which are crucial for recognition of PC on apoptotic cell surface and other neo-epitopes. Conclusion Anti-PC IgG1 is negatively associated with disease activity, and disease damage in SLE, but the negative association with CVD is also dependent on confounding risk factors. One potential underlying mechanism could be increased clearance of dead cells. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Swedish Heart and Lung Foundation, Reumatikerfonden

Immunoglobulin G1 Antibodies Against Phosphorylcholine Are Associated With Protection in Systemic Lupus Erythematosus and Atherosclerosis: Potential Underlying Mechanisms.

ObjectiveImmunoglobulin M antibodies against phosphorylcholine (anti‐PCs) may be protective in atherosclerosis, cardiovascular disease (CVD), and systemic lupus erythematosus (SLE). We study immunoglobulin G1 (IgG1) and immunoglobulin G2 (IgG2) anti‐PCs, with a focus on atherosclerosis and SLE.MethodsWe determined anti‐PCs by using the enzyme‐linked immunosorbent assay in 116 patients with SLE and 110 age‐ and sex‐matched controls. For functional studies, we used three in‐house–generated, fully human monoclonal IgG1 anti‐PCs (A01, D05, and E01). Apoptosis was induced in Jurkat T cells and preincubated with A01, D05, E01, or IgG1 isotype control, and effects on efferocytosis by human macrophages were studied. Anti‐PC peptide/protein characterization was determined using a proteomics de novo sequencing approach.ResultsIgG1, but not IgG2, anti‐PC levels were higher among patients with SLE (P = 0.02). IgG1 anti‐PCs were negatively associated with Systemic Lupus International Collaborating Clinics (SLICC) damage index and Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) scores (odds ratio [OR]: 2.978 [confidence interval (CI): 0.876‐10.098] and OR: 5.108 [CI 1.3‐20.067], respectively) and negatively associated with CVD, atherosclerotic plaques, and echolucent plaques (potentially vulnerable plaques), but the association for the two former was not significant after controlling for confounders. D05 had a maximum effect on macrophage efferocytosis efficiency, followed by A01 and E01. The monoclonal antibodies showed differential binding specificity to PC and PC‐associated neoepitopes. A peptide analysis showed a difference in the complementarity‐determining region 3 of the three IgG1 anti‐PC clones that are crucial for recognition of PC on apoptotic cell surfaces and other neoepitopes.ConclusionIgG1 anti‐PCs are negatively associated with disease activity and disease damage in SLE, but the negative association with CVD is also dependent on confounding risk factors. One potential underlying mechanism could be increased clearance of dead cells.

Therapeutic anti‐P2X4 receptor scFv antibody for chronic neuropathic pain

The P2X purinoceptor 4 (P2X4) plays a crucial role in signaling leading to chronic inflammatory and neuropathic pains, thus, the P2X4 receptor provides a new potential therapeutic target for their treatment. We have generated a panel of mouse single‐chain Fv‐antibodies (scFvs) recognizing extracellular peptide of P2X4 using cell‐free ribosome display, for use to pain‐signalling neurons. A mouse scFv library was generated, and ribosome display was applied for anti‐P2X4 scFv recombinant antibody selection. After three rounds of bio‐panning, a panel of recombinant antibodies was isolated followed by ELISA, cross reactivity analysis, western blotting and immunofluorescence staining. Anti‐P2X4 scFv clones with high specificity and affinity were successfully selected. Indirect ELISA assay revealed differential P2X4 peptide binding capability and specificity for three scFvs, with no reaction to the negative control anti‐Zika scFv 7‐2. The panning process was efficient in selecting clones of high affinity, as was evident from affinity differences of the scFvs: scFv95 and scFv12 had the highest and second highest affinities, respectively, while scFv103 had lower affinity. The highest affinity P2X4 receptor scFvs (12 and 95) were selected for in vivo behavior testing in the Foramen Rotundum Inflammatory Constriction Trigeminal Infraorbital Nerve injury (FRICT‐ION) Model. Single dose intraperitoneal injection (4 mg/kg, 100 μl) reversed mechanical allodynia and anxiety in mice with long standing neuropathic pain (8–10 weeks). Reversal to naïve baseline within one week was maintained for more than 10 weeks post‐treatment at experiment end. Continued reversal was maintained at baseline for >20 weeks in a second cohort. The Western blot of the medullary brainstem provides evidence of brain penetration of both these scFv antibodies that reduce hypersensitivity and anxiety behaviors. We also performed whole‐cell patch clamp recordings from dissociated trigeminal ganglia neurons of nerve‐injured vs naïve mice and determined the effect of P2X4 receptor scFvs on neuronal excitability. These data provide support for pursuit of P2X4 receptor scFvs as translational therapy for pain relief.Support or Funding Information1. GRANT FUNDING NIH NIDCR R21 DE028096 2. Start‐up Funds to Dr. Durvasula 3. Startup Funds to Dr. Westlund

Chromatin accessibility plays a key role in selective targeting of Hox proteins

BackgroundHox transcription factors specify segmental diversity along the anterior-posterior body axis in metazoans. While the different Hox family members show clear functional specificity in vivo, they all show similar binding specificity in vitro and a satisfactory understanding of in vivo Hox target selectivity is still lacking.ResultsUsing transient transfection in Kc167 cells, we systematically analyze the binding of all eight Drosophila Hox proteins. We find that Hox proteins show considerable binding selectivity in vivo even in the absence of canonical Hox cofactors Extradenticle and Homothorax. Hox binding selectivity is strongly associated with chromatin accessibility, being highest in less accessible chromatin. Individual Hox proteins exhibit different propensities to bind less accessible chromatin, and high binding selectivity is associated with high-affinity binding regions, leading to a model where Hox proteins derive binding selectivity through affinity-based competition with nucleosomes. Extradenticle/Homothorax cofactors generally facilitate Hox binding, promoting binding to regions in less accessible chromatin but with little effect on the overall selectivity of Hox targeting. These cofactors collaborate with Hox proteins in opening chromatin, in contrast to the pioneer factor, Glial cells missing, which facilitates Hox binding by independently generating accessible chromatin regions.ConclusionsThese studies indicate that chromatin accessibility plays a key role in Hox selectivity. We propose that relative chromatin accessibility provides a basis for subtle differences in binding specificity and affinity to generate significantly different sets of in vivo genomic targets for different Hox proteins.

Specificity of Escherichia coli Heat-Labile Enterotoxin Investigated by Single-Site Mutagenesis and Crystallography.

Diarrhea caused by enterotoxigenic Escherichia coli (ETEC) is one of the leading causes of mortality in children under five years of age and is a great burden on developing countries. The major virulence factor of the bacterium is the heat-labile enterotoxin (LT), a close homologue of the cholera toxin. The toxins bind to carbohydrate receptors in the gastrointestinal tract, leading to toxin uptake and, ultimately, to severe diarrhea. Previously, LT from human- and porcine-infecting ETEC (hLT and pLT, respectively) were shown to have different carbohydrate-binding specificities, in particular with respect to N-acetyllactosamine-terminating glycosphingolipids. Here, we probed 11 single-residue variants of the heat-labile enterotoxin with surface plasmon resonance spectroscopy and compared the data to the parent toxins. In addition we present a 1.45 Å crystal structure of pLTB in complex with branched lacto-N-neohexaose (Galβ4GlcNAcβ6[Galβ4GlcNAcβ3]Galβ4Glc). The largest difference in binding specificity is caused by mutation of residue 94, which links the primary and secondary binding sites of the toxins. Residue 95 (and to a smaller extent also residues 7 and 18) also contribute, whereas residue 4 shows no effect on monovalent binding of the ligand and may rather be important for multivalent binding and avidity.

Differential CLE peptide perception by plant receptors implicated from structural and functional analyses of TDIF-TDR interactions.

Tracheary Element Differentiation Inhibitory Factor (TDIF) belongs to the family of post-translationally modified CLE (CLAVATA3/embryo surrounding region (ESR)-related) peptide hormones that control root growth and define the delicate balance between stem cell proliferation and differentiation in SAM (shoot apical meristem) or RAM (root apical meristem). In Arabidopsis, Tracheary Element Differentiation Inhibitory Factor Receptor (TDR) and its ligand TDIF signaling pathway is involved in the regulation of procambial cell proliferation and inhibiting its differentiation into xylem cells. Here we present the crystal structures of the extracellular domains (ECD) of TDR alone and in complex with its ligand TDIF resolved at 2.65 Ǻ and 2.75 Ǻ respectively. These structures provide insights about the ligand perception and specific interactions between the CLE peptides and their cognate receptors. Our in vitro biochemical studies indicate that the interactions between the ligands and the receptors at the C-terminal anchoring site provide conserved binding. While the binding interactions occurring at the N-terminal anchoring site dictate differential binding specificities between different ligands and receptors. Our studies will open different unknown avenues of TDR-TDIF signaling pathways that will enhance our knowledge in this field highlighting the receptor ligand interaction, receptor activation, signaling network, modes of action and will serve as a structure function relationship model between the ligand and the receptor for various similar leucine-rich repeat receptor-like kinases (LRR-RLKs).

The sequence, structural, and functional diversity within a protein family and implications for specificity and safety: The case for ETX_MTX2 insecticidal proteins

The need for sustainable insect pest control is driving the investigation and discovery of insecticidal proteins outside of the typical 3-domain Cry protein family from Bacillus thuringiensis (Bt). Examples include Cry35 and Cry51 that belong to protein families (Toxin_10, ETX_MTX2) sharing a common β-pore forming structure and function with known mammalian toxins such as epsilon toxin (ETX). Although β-pore forming proteins are related to mammalian toxins, there are key differences in sequence and structure that lead to organism specificity that is useful in the weight-of-evidence approach for safety assessment. Despite low overall amino acid sequence identity among ETX_MTX2 proteins, sequence and structural similarities are found in the tail region responsible for the shared oligomerization and pore formation functions (causing the “relatedness”). Conversely, most of the sequence and structural diversity is located in the head region that is likely responsible for differential receptor binding and target species specificity (e.g., insecticidal vs. mammalian). Therefore, inclusion of a domain-based protein characterization approach that includes bioinformatic and functional comparisons of conserved and diverse domains will enhance the overall weight of evidence safety assessment of proteins including recently reported Cry51 protein variants (Cry51Aa1, Cry51Aa2, and Cry51Aa2.834_16).

CLEC18 family are novel C-type lectins with differential binding specificity to glycans and TLR ligands

Abstract The human C-type lectin 18 (clec18) gene cluster, which contains clec18a, clec18b and clec18c three loci, is located in human chromosome 16q22. Even though the amino acid sequences of CLEC18A, CLEC18B, and CLEC18C are almost identical, several amino acid residues located in the C-type lectin-like domain (CTLD) and the Sperm-Coating Protein/Tpx-1/Ag5/PR-1/Sc7 (SCP/TAPS) domain, also known as the cysteine-rich secretory proteins/antigen 5/pathogenesis-related 1 proteins (CAP) domain, are distinct from each other. Genotyping by real-time PCR and sequencing further shows the presence of multiple alleles in clec18a/b/c loci. Flow cytometry analysis demonstrates that CLEC18 (CLEC18A, B and C) are expressed abundantly in human peripheral blood cells. Moreover, CLEC18 expression is further upregulated when monocytes differentiate into macrophages and dendritic cells (DCs). Immunofluorescence staining reveals that CLEC18 are localized in the endoplasmic reticulum (ER), Golgi apparatus, and endosome. Interestingly, CLEC18 are also detectable in human sera and culture supernatants from primary cells and 293T cells overexpressing CLEC18. Moreover, CLEC18 bind polysaccharide in Ca2+–independent manner, and the amino acid residues S/R339 and D/N421 in CTLD domain contribute to their differential binding abilities to polysaccharides isolated from Ganoderma lucidum (GLPS-F3). The S339 (CLEC18A) and R339 (CLEC18A-1) displayed differential binding affinity to TLR ligands. Their roles to determine host responses to virus infection are under intensive investigation now.

Structural Characterization of Substrate Transport Selectivity of the SLC13 Family of Na+/Dicarboxylate Cotransporters

Members of the sodium dependent SLC13 transporter family mediate the transport of di and tricarboxylates of the citric acid cycle into cells. SLC13 transporters such as NaCT (SLC13A5) have been implicated in lifespan extension and resistance to high fat diets and are therefore emerging drug targets for aging and metabolic disorders. Here, we combine various computational and experimental methods to improve our understanding of the structural components that are important for substrate specificity in this physiologically important family. We first used homology modeling, site-directed mutagenesis and transport assays to characterize the human NaDC3 (SLC13A3) and identify key residues involved in substrate and Na+ binding. We then expanded our study and generated homology models for various SLC13 transporters from the human and mouse. Virtual screening of various small molecule libraries followed by experimental testing revealed nine previously unknown inhibitors of multiple members of the familly. Ligand optimization showed that differential ligand binding specificity among SLC13 family members is largely determined by the length of the carbon chain and the size of the ligands. Additionally, we identified two key positions that define a unique binding site for each SLC13 member. Our findings rationalize substrate specificity in the SLC13 family and provide a framework for the development of more potent inhibitors.

VASP-E: specificity annotation with a volumetric analysis of electrostatic isopotentials.

Algorithms for comparing protein structure are frequently used for function annotation. By searching for subtle similarities among very different proteins, these algorithms can identify remote homologs with similar biological functions. In contrast, few comparison algorithms focus on specificity annotation, where the identification of subtle differences among very similar proteins can assist in finding small structural variations that create differences in binding specificity. Few specificity annotation methods consider electrostatic fields, which play a critical role in molecular recognition. To fill this gap, this paper describes VASP-E (Volumetric Analysis of Surface Properties with Electrostatics), a novel volumetric comparison tool based on the electrostatic comparison of protein-ligand and protein-protein binding sites. VASP-E exploits the central observation that three dimensional solids can be used to fully represent and compare both electrostatic isopotentials and molecular surfaces. With this integrated representation, VASP-E is able to dissect the electrostatic environments of protein-ligand and protein-protein binding interfaces, identifying individual amino acids that have an electrostatic influence on binding specificity. VASP-E was used to examine a nonredundant subset of the serine and cysteine proteases as well as the barnase-barstar and Rap1a-raf complexes. Based on amino acids established by various experimental studies to have an electrostatic influence on binding specificity, VASP-E identified electrostatically influential amino acids with 100% precision and 83.3% recall. We also show that VASP-E can accurately classify closely related ligand binding cavities into groups with different binding preferences. These results suggest that VASP-E should prove a useful tool for the characterization of specific binding and the engineering of binding preferences in proteins.