N-terminal Cysteine-rich Domain Research Articles

SIRT4 as a novel interactor and candidate suppressor of C-RAF kinase in MAPK signaling.

Cellular responses leading to development, proliferation, and differentiation depend on RAF/MEK/ERK signaling, which integrates and amplifies signals from various stimuli for downstream cellular responses. C-RAF activation has been reported in many types of tumor cell proliferation and developmental disorders, necessitating the discovery of potential C-RAF protein regulators. Here, we identify a novel and specific protein interaction between C-RAF among the RAF kinase paralogs, and SIRT4 among the mitochondrial sirtuin family members SIRT3, SIRT4, and SIRT5. Structurally, C-RAF binds to SIRT4 through the N-terminal cysteine-rich domain, whereas SIRT4 predominantly requires the C-terminus for full interaction with C-RAF. Interestingly, SIRT4 specifically interacts with C-RAF in a pre-signaling inactive (serine 259-phosphorylated) state. Consistent with this finding, the expression of SIRT4 in HEK293 cells results in an up-regulation of pS259-C-RAF levels and a concomitant reduction in MAPK signaling as evidenced by strongly decreased phospho-ERK signals. Thus, we propose an additional extra-mitochondrial function of SIRT4 as a cytosolic tumor suppressor of C-RAF-MAPK signaling, besides its metabolic tumor suppressor role of glutamate dehydrogenase and glutamate levels in mitochondria.

Characteristics of Shisa Family Genes in Zebrafish.

Shisa represents a type of single-transmembrane adaptor protein containing an N-terminal cysteine-rich domain and a proline-rich C-terminal region. Nine shisa subfamily genes have been proposed in most vertebrates; however, some might be species-specific. The number of shisa genes present in zebrafish remains unclear. This study aimed to investigate the evolutionary relationships among shisa family genes in zebrafish (TU strain) using phylogenetic and syntenic analyses. The function of shisa-2 was preliminarily examined via CRISPR/Cas13d-mediated knockdown. Following identification in zebrafish, 10 shisa family genes, namely shisa-1, 2, 3, 4, 5, 6, 7, 8, 9a, and 9b, were classified into three main clades and six subclades. Their encoding proteins contained a cysteine-rich N-terminal domain and a proline-rich C-terminal region containing different motifs. A specific syntenic block containing atp8a2 and shisa-2 was observed to be conserved across all species. Furthermore, all these genes were expressed during embryogenesis. Shisa-2 was expressed in the presomitic mesoderm, somites, and so on. Shisa-2 was identified as a regulator of the expression of the somite formation marker mesp-ab. Overall, our study provides new insights into the evolution of shisa family genes and the control of shisa-2 over the convergent extension cells of somitic precursors in zebrafish.

Structural insights into pathogenic mechanism of hypohidrotic ectodermal dysplasia caused by ectodysplasin A variants

EDA is a tumor necrosis factor (TNF) family member, which functions together with its cognate receptor EDAR during ectodermal organ development. Mutations of EDA have long been known to cause X‐linked hypohidrotic dysplasia in humans characterized by primary defects in teeth, hair and sweat glands. However, the structural information of EDA interaction with EDAR is lacking and the pathogenic mechanism of EDA variants is poorly understood. Here, we report the crystal structure of EDA C-terminal TNF homology domain bound to the N-terminal cysteine-rich domains of EDAR. Together with biochemical, cellular and mouse genetic studies, we show that different EDA mutations lead to varying degrees of ectodermal developmental defects in mice, which is consistent with the clinical observations on human patients. Our work extends the understanding of the EDA signaling mechanism, and provides important insights into the molecular pathogenesis of disease-causing EDA variants.

Abstract 1856: Investigating the role of the N-terminal cysteine-rich domain of violaxanthin de-epoxidase like -2 (VDL-2) in redox sensing in diatoms

Abstract 1856: Investigating the role of the N-terminal cysteine-rich domain of violaxanthin de-epoxidase like -2 (VDL-2) in redox sensing in diatoms

Abstract 2270: Membrane organization of KRas4B with regulatory domain of Raf-1

Abstract A major pathway in RAS-driven cell proliferation is the MAPK (Raf/MEK/ERK) pathway. In cancer, membrane-anchored, proximally located, oncogenic Ras isoforms promote Raf dimerization and fully activate MAPK signaling. Among Ras isoforms, KRas4B is frequently mutated in cancer. In the MAPK pathway, active KRas4B dimer or nanocluster can lead to dimerization of Raf kinase domain enhancing the affinity of the KRas4B-Raf interaction. Recently, we modeled the binary KRas4B-Raf-1 complex and its quaternary assembly with the regulatory domain of Raf-1 containing the Ras binding domain (RBD) and cysteine-rich domain (CRD), and suggested that Raf-1 CRD bolsters the high-affinity interaction of Raf-1 with KRas4B by reducing the KRas4B-RBD fluctuations. For the quaternary assembly, recent in silico studies suggested that KRas4B dimer is aligned through the α3 and α4 helical interface, not the α4 and α5 helical interface due to hindrance to the membrane contact of Raf-1 CRD. The Raf-1 regulatory domain contains the N-terminal region (residues 1-55), RBD, and CRD. However, previous modeling efforts were conducted for the KRas4B-Raf-1 complex in the absence of the N-terminal region. Here, computational studies were performed for the KRas4B-Raf-1 complex with full sequence of Raf-l regulatory domain interacting with monomeric and dimeric KRas4B-GTP at the membrane. Increasing experimental evidences suggested that the N-terminal region of Raf plays a significant role in the Ras interaction and the membrane anchorage of Raf. The membrane interaction of isolated N-terminal segment was investigated as the first step toward the complex model construction. Since oncogenic KRas4B mutants form dimers/nanoclusters at the membrane microdomains that provide the signaling platform for Raf, KRas4B in different mutation states interacting with the membrane microdomains with different lipid composition was also studied. The presence of the N-terminal segment of Raf-1 regulatory domain at the membrane can enhance the stability of KRas4B-Raf-1 and thereby relieving Raf's autoinhibition toward a kinase domain-accessible state, providing a complete mechanistic picture of the KRas4B-Raf-1 interaction. Our recent work uncovers the mechanism of Raf-1 activation by KRas4B at atomic resolution and highlight how this may help in elucidating vital mechanistic questions in Ras biology. Funded by Frederick National Laboratory for Cancer Research, National Institutes of Health, under contract HHSN261200800001E. Citation Format: Hyunbum Jang, Mingzhen Zhang, Ruth Nussinov. Membrane organization of KRas4B with regulatory domain of Raf-1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2270.

Understanding the binding affinities between SFRP1 CRD , SFRP1 Netrin , Wnt 5B and frizzled receptors 2, 3 and 7 using MD simulations

cWnt-signalling plays a crucial role in stem cell maintenance and tissue homeostasis. Secreted frizzled-related proteins(SFRP), Wnt inhibitors consist of the N-terminal cysteine rich domain(CRD) and the C-terminal netrin(NTR) domain. SFRP1 binds to the Wnt ligands and frizzled receptors(FZ) either through its SFRP1CRD or through its SFRP1Netrin domains; however, very little is known on these binding affinities. Here, we attempted to understand the interactions and binding affinities of SFRP1-Wnt5B, SFRP1-FZ(2, 3 & 7) and Wnt5B-FZ(2, 3 & 7) that are mainly expressed in murine hair follicle stem cells. SFRP1CRD, SFRP1Netrin, Wnt5B and FZ(2, 3 & 7) structures were built using homology modelling, followed by their molecular dynamics simulations. SFRP1CRD showed lower fluctuation when in complex with FZ2, FZ3 and FZ7 and Wnt5B as compared to SFRP1Netrin using RMSF and RMSD. However, free energy showed SFRP1Netrin was energetically more stable than SFRP1CRD. SFRP1Netrin formed more number of interactions with FZ as compared to SFRP1CRD. Importantly, SFRP1Netrin favoured binding to the FZ receptors(FZ3 > FZ7 > FZ2) as compared to Wnt5B ligand. Conversely, the SFRP1CRD showed more affinity towards the Wnt5B ligand as compared to FZ receptors. Wnt5B showed the best binding affinity with FZ3 followed by SFRP1CRD and SFRP1Netrin. Therefore, SFRP1Netrin can bind to the FZ3 with higher binding affinity and may inhibit non-canonical Wnt-signalling pathway. Our study provides the comprehensive information on the binding affinities among the Wnt5B, SFRP1CRD/Netrin and FZ(2, 3 & 7). Thus, this information might also help in designing novel strategies to inhibit aberrant Wnt-signalling. Communicated by Ramaswamy H. Sarma

The cryo-EM structure of the endocytic receptor DEC-205

DEC-205 (CD205), a member of the macrophage mannose receptor protein family, is the prototypic endocytic receptor of dendritic cells, whose ligands include phosphorothioated cytosine–guanosine oligonucleotides, a motif often seen in bacterial or viral DNA. However, despite growing biological and clinical significance, little is known about the structural arrangement of this receptor or any of its family members. Here, we describe the 3.2 Å cryo-EM structure of human DEC-205, thereby illuminating the structure of the mannose receptor protein family. The DEC-205 monomer forms a compact structure comprising two intercalated rings of C-type lectin-like domains, where the N-terminal cysteine-rich and fibronectin domains reside at the central intersection. We establish a pH-dependent oligomerization pathway forming tetrameric DEC-205 using solution-based techniques and ultimately solved the 4.9 Å cryo-EM structure of the DEC-205 tetramer to identify the unfurling of the second lectin ring which enables tetramer formation. Furthermore, we suggest the relevance of this oligomerization pathway within a cellular setting, whereby cytosine–guanosine binding appeared to disrupt this cell-surface oligomer. Accordingly, we provide insight into the structure and oligomeric assembly of the DEC-205 receptor.

Disulfide-Linked Peptides for Blocking BTLA/HVEM Binding.

Immune checkpoints are crucial in the maintenance of antitumor immune responses. The activation or blockade of immune checkpoints is dependent on the interactions between receptors and ligands; such interactions can provide inhibitory or stimulatory signals, including the enhancement or suppression of T-cell proliferation, differentiation, and/or cytokine secretion. B-and T-lymphocyte attenuator (BTLA) is a lymphoid-specific cell surface receptor which is present on T-cells and interacts with herpes virus entry mediator (HVEM), which is present on tumor cells. The binding of HVEM to BTLA triggers an inhibitory signal which attenuates the immune response. This feature is interesting for studying the molecular interactions between HVEM and BTLA, as they may be targeted for novel immunotherapies. This work was based on the crystal structure of the BTLA/HVEM complex showing that BTLA binds the N-terminal cysteine-rich domain of HVEM. We investigated the amino acid sequence of HVEM and used molecular modeling methods to develop inhibitors of the BTLA/HVEM interaction. We synthesized novel compounds and determined their ability to interact with the BTLA protein and inhibit the formation of the BTLA/HVEM complex. Our results suggest that the HVEM (14–39) peptide is a potent inhibitor of the formation of the BTLA/HVEM protein complex.

Regulatory Mechanism of the Atypical AP-1-Like Transcription Factor Yap1 in Cryptococcus neoformans.

AP-1-like transcription factors play evolutionarily conserved roles as redox sensors in eukaryotic oxidative stress responses. In this study, we aimed to elucidate the regulatory mechanism of an atypical yeast AP-1-like protein, Yap1, in the stress response and virulence of Cryptococcus neoformansYAP1 expression was induced and involved not only by oxidative stresses, such as H2O2 and diamide, but also by other environmental stresses, such as osmotic and membrane-destabilizing stresses. Yap1 was distributed throughout both the cytoplasm and the nucleus under basal conditions and more enriched within the nucleus in response to diamide but not to other stresses. Deletion of the C-terminal cysteine-rich domain (c-CRD), where the nuclear export signal resides, increased nuclear enrichment of Yap1 under basal conditions and altered resistance to oxidative stresses but did not affect the role of Yap1 in other stress responses and cellular functions. As a potential upstream regulator of Yap1, we discovered that Mpk1 is positively involved, but Hog1 is mostly dispensable. Pleiotropic roles for Yap1 in diverse biological processes were supported by transcriptome data showing that 162 genes are differentially regulated by Yap1, with further analysis revealing that Yap1 promotes cellular resistance to toxic cellular metabolites produced during glycolysis, such as methylglyoxal. Finally, we demonstrated that Yap1 plays a minor role in the survival of C. neoformans within hosts.IMPORTANCE The human meningitis fungal pathogen, Cryptococcus neoformans, contains the atypical yeast AP-1-like protein Yap1. Yap1 lacks an N-terminal cysteine-rich domain (n-CRD), which is present in other fungal Yap1 orthologs, but has a C-terminal cysteine-rich domain (c-CRD). However, the role of c-CRD and its regulatory mechanism remain unknown. Here, we report that Yap1 is transcriptionally regulated in response to oxidative, osmotic, and membrane-destabilizing stresses partly in an Mpk1-dependent manner, supporting its role in stress resistance. The c-CRD domain contributed to the role of Yap1 only in resistance to certain oxidative stresses and azole drugs but not in other cellular functions. Yap1 has a minor role in the survival of C. neoformans in a murine model of systemic cryptococcosis.

Structure of human cytomegalovirus UL144, an HVEM orthologue, bound to the B and T cell lymphocyte attenuator

Human cytomegalovirus (HCMV) is a β-herpesvirus that has co-evolved with the host immune system to establish lifelong persistence. HCMV encodes many immunomodulatory molecules, including the glycoprotein UL144. UL144 is a structural mimic of the tumor necrosis factor receptor superfamily member HVEM (herpesvirus entry mediator), which binds to the various ligands LIGHT, LTα, BTLA, CD160, and gD. However, in contrast to HVEM, UL144 only binds BTLA, inhibiting T-cell activation. Here, we report the crystal structure of the UL144-BTLA complex, revealing that UL144 utilizes residues from its N-terminal cysteine-rich domain 1 (CRD1) to interact uniquely with BTLA. The shorter CRD2 loop of UL144 also alters the relative orientation of BTLA binding with both N-terminal CRDs. By employing structure-guided mutagenesis, we have identified a mutant of BTLA (L123A) that interferes with HVEM binding but preserves UL144 interactions. Furthermore, our results illuminate structural differences between UL144 and HVEM that explain its binding selectivity and highlight it as a suitable scaffold for designing superior, immune inhibitory BTLA agonists.

Cloning and expression of ATP N-glycosidase from the freshwater sponge Ephydatia muelleri

Previously we had discovered unusual enzymatic activity in the marine sponge Axinella polypoides, ATP N-glycosidase (Reintamm et al., 2003). We show here that the Ephydatia muelleri mRNA encoding protein with PNP_UDP_1 (phosphorylase superfamily) signature is the secreted ATP N-glycosidase. The functionality of the protein was established by recombinant expression in Pichia pastoris. In addition to the enzymatic domain, the full-length protein contains the N-terminal cysteine-rich domain belonging to the subfamily SCP_HrTT-1 (cd05559) of the SCP (sperm coating protein) superfamily (cl00133).

SURGE complex of Plasmodium falciparum in the rhoptry-neck (SURFIN4.2-RON4-GLURP) contributes to merozoite invasion.

Plasmodium falciparum invasion into red blood cells (RBCs) is a complex process engaging proteins on the merozoite surface and those contained and sequentially released from the apical organelles (micronemes and rhoptries). Fundamental to invasion is the formation of a moving junction (MJ), a region of close apposition of the merozoite and the RBC plasma membranes, through which the merozoite draws itself before settling into a newly formed parasitophorous vacuole (PV). SURFIN4.2 was identified at the surface of the parasitized RBCs (pRBCs) but was also found apically associated with the merozoite. Using antibodies against the N-terminus of the protein we show the presence of SURFIN4.2 in the neck of the rhoptries, its secretion into the PV and shedding into the culture supernatant upon schizont rupture. Using immunoprecipitation followed by mass spectrometry we describe here a novel protein complex we have named SURGE where SURFIN4.2 forms interacts with the rhoptry neck protein 4 (RON4) and the Glutamate Rich Protein (GLURP). The N-terminal cysteine-rich–domain (CRD) of SURFIN4.2 mediates binding to the RBC membrane and its interaction with RON4 suggests its involvement in the contact between the merozoite apex and the RBC at the MJ. Supporting this suggestion, we also found that polyclonal antibodies to the extracellular domain (including the CRD) of SURFIN4.2 partially inhibit merozoite invasion. We propose that the formation of the SURGE complex participates in the establishment of parasite infection within the PV and the RBCs.

Structural and functional analysis of Dickkopf 4 (Dkk4): New insights into Dkk evolution and regulation of Wnt signaling by Dkk and Kremen proteins

Dickkopf (Dkk) family proteins are important regulators of Wnt signaling pathways, which play key roles in many essential biological processes. Here, we report the first detailed structural and dynamics study of a full-length mature Dkk protein (Dkk4, residues 19–224), including determination of the first atomic-resolution structure for the N-terminal cysteine-rich domain (CRD1) conserved among Dkk proteins. We discovered that CRD1 has significant structural homology to the Dkk C-terminal cysteine-rich domain (CRD2), pointing to multiple gene duplication events during Dkk family evolution. We also show that Dkk4 consists of two independent folded domains (CRD1 and CRD2) joined by a highly flexible, nonstructured linker. Similarly, the N-terminal region preceding CRD1 and containing a highly conserved NXI(R/K) sequence motif was shown to be dynamic and highly flexible. We demonstrate that Dkk4 CRD2 mediates high-affinity binding to both the E1E2 region of low-density lipoprotein receptor–related protein 6 (LRP6 E1E2) and the Kremen1 (Krm1) extracellular domain. In contrast, the N-terminal region alone bound with only moderate affinity to LRP6 E1E2, consistent with binding via the conserved NXI(R/K) motif, but did not interact with Krm proteins. We also confirmed that Dkk and Krm family proteins function synergistically to inhibit Wnt signaling. Insights provided by our integrated structural, dynamics, interaction, and functional studies have allowed us to refine the model of synergistic regulation of Wnt signaling by Dkk proteins. Our results indicate the potential for the formation of a diverse range of ternary complexes comprising Dkk, Krm, and LRP5/6 proteins, allowing fine-tuning of Wnt-dependent signaling.

G protein βγ subunits directly interact with and activate phospholipase CΕ

Phospholipase C (PLC) enzymes hydrolyze membrane phosphatidylinositol 4,5 bisphosphate (PIP2) and regulate Ca2+ and protein kinase signaling in virtually all mammalian cell types. Chronic activation of the PLCϵ isoform downstream of G protein-coupled receptors (GPCRs) contributes to the development of cardiac hypertrophy. We have previously shown that PLCϵ-catalyzed hydrolysis of Golgi-associated phosphatidylinositol 4-phosphate (PI4P) in cardiac myocytes depends on G protein βγ subunits released upon stimulation with endothelin-1. PLCϵ binds and is directly activated by Ras family small GTPases, but whether they directly interact with Gβγ has not been demonstrated. To identify PLCϵ domains that interact with Gβγ, here we designed various single substitutions and truncations of WT PLCϵ and tested them for activation by Gβγ in transfected COS-7 cells. Deletion of only a single domain in PLCϵ was not sufficient to completely block its activation by Gβγ, but blocked activation by Ras. Simultaneous deletion of the C-terminal RA2 domain and the N-terminal CDC25 and cysteine-rich domains completely abrogated PLCϵ activation by Gβγ, but activation by the GTPase Rho was retained. In vitro reconstitution experiments further revealed that purified Gβγ directly interacts with a purified fragment of PLCϵ (PLCϵ-PH-RA2) and increases PIP2 hydrolysis. Deletion of the RA2 domain decreased Gβγ binding and eliminated Gβγ stimulation of PIP2 hydrolysis. These results provide first evidence that Gβγ directly interacts with PLCϵ and yield insights into the mechanism by which βγ subunits activate PLCϵ.

The crystal structure of full-length Sizzled from Xenopus laevis yields insights into Wnt-antagonistic function of secreted Frizzled-related proteins

The Wnt-signaling pathway is crucial to cell proliferation, differentiation, and migration. The secreted Frizzled-related proteins (sFRPs) represent the largest family of secreted Wnt inhibitors. However, their function in antagonizing Wnt signaling has remained somewhat controversial. Here, we report the crystal structure of Sizzled from Xenopus laevis, the first full-length structure of an sFRP. Tethered by an inter-domain disulfide bond and a linker, the N-terminal cysteine-rich domain (CRD) and the C-terminal netrin-like domain (NTR) of Sizzled are arranged in a tandem fashion, with the NTR domain occluding the groove of CRD for Wnt accessibility. A Dual-Luciferase assay demonstrated that removing the NTR domain and replacing the CRD groove residues His-116 and His-118 with aromatic residues may significantly enhance antagonistic function of Sizzled in inhibiting Wnt3A signaling. Sizzled is a monomer in solution, and Sizzled CRD exhibited different packing in the crystal, suggesting that sFRPs do not have a conserved CRD dimerization mode. Distinct from the canonical NTR domain, the Sizzled NTR adopts a novel α/β folding with two perpendicular helices facing the central mixed β-sheet. The subgroup of human sFRP1/2/5 and Sizzled should have a similar NTR domain that features a highly positively charged region, opposite the NTR-CRD interface, suggesting that the NTR domain in human sFRPs, at least sFRP1/2/5, is unlikely to bind to Wnt but is likely involved in biphasic Wnt signaling modulation. In summary, the Sizzled structure provides the first insights into how the CRD and the NTR domains relate to each other for modulating Wnt-antagonistic function of sFRPs.

Novel domain-targeted anti-Trop-2 monoclonal antibodies to elicit therapeutic synergy against multiple human cancers.

e14002 Background: Trop-2 is a calcium signal transducer that drives tumor cell growth. Trop-2 is overexpressed in the majority of carcinomas, where it associates with worse prognosis. The Trop-2 extracellular domain contains a N-terminal cysteine-rich globular domain followed by a stem-like cysteine-less region that connects to the transmembrane domain. Trop-2 engages in homophylic interactions between adjacent cells and interacts with tight-junction proteins, which may severely affect accessibility by therapeutic monoclonal antibodies (mAb). Available anti-Trop-2 mAb mostly recognize a single immunodominant epitope between the globular and stem regions, and have limited or no therapeutic efficacy. In order to untap the potential of anti-Trop-2 immunotherapy we generated novel anti-Trop-2 mAb with tailored specificity towards the globular versus stem regions. Methods: Balb-C mice were immunized with soluble human Trop-2 produced in human 293 and murine L cell lines and in baculovirus expression system. Domain-targeted anti-Trop-2 mAb were selected by flow cytometry using live 293 transfectants.Therapeutic potential was assessed in human cancer xenografts in murine models. mAb mode of action was investigated by Western blot, live-cell imaging and in vitro ADCC and cancer cell growth inhibition assays. Results: mAb were identified that were differentially directed against the Trop-2 stem versus globular regions. These mAb efficiently bound Trop-2 expressing cancer cells and inhibited cell growth in vitro. In vivo, naked anti-globular OX-G64 and anti-stem OX-S55 mAb were most effective in inhibiting the growth of colon, ovary, breast and prostate cancers as xenografts in nude mice. NSG mice and in vitro mechanism profiling indicated efficient ADCC, together with efficient internalization for ADC development. Differential efficacy for established tumors versus isolated-cell models of metastatic dissemination was shown. Most remarkably, OX-G64/OX-S55 co-administration demonstrated sinergic growth inhibition in vivo. Conclusions: The OX-G64 and OX-S55 anti-Trop-2 mAb are novel domain-targeted, sinergic therapeutic mAb for game-changing anti-cancer immunotherapy.

Structural Characterization of Human Pulmonary Surfactant Protein SP-D by Atomic Force Microscopy

Pulmonary surfactant protein D (SP-D), a C-type (Ca2+-dependent) lectin, belongs to the collectin family. It is found mainly in lung surfactant, participating in the innate immune defense of the lungs.SP-D monomer contains four structural domains: an N-terminus domain, a collagen region, an α-helical coiled-coil neck and a C-terminus carbohydrate recognition domain (CRD). Monomers form trimers through folding of the collagenous region into triple helices and the assembly of a coiled-coil bundle of α-helices in the neck region. These trimers are stabilized by two disulfide bonds in the cysteine-rich N-terminal domain. Trimers associate into higher order oligomers whose size and conformation is sensitive to environmental factors and the conditions during purification and storage. Despite extensive studies carried out to characterize the oligomerization process of SP-D, the pathway and type of interactions involved in the formation of large oligomers, are not clearly understood. In the current study, a structural characterization of recombinant human SP-D (rhSP-D), produced in mammalian CHO cells, has been carried out by Atomic Force Microscopy (AFM) and electrophoresis. The goal has been the determination of the distribution of oligomeric forms, the exploration of the possible oligomerization pathway and the description of the conformational diversity of rhSP-D. AFM experiments revealed that rhSP-D is a mixture of trimers, hexamers, dodecamers, and larger oligomeric species identified as “fuzzy balls”, with the most abundant structure being the dodecamer under the conditions of these experiments. We have developed a cross-linking protocol to detect the presence of SP-D dodecameric forms by PAGE-SDS, in which dodecamer is only visualized after chemical crosslinking and in the presence of denaturing agents, indicating the importance of hydrophobic interactions in dodecamer formation.

A molecular dynamics approach to identify an oxysterol-based hedgehog pathway inhibitor

BackgroundMultiple oxysterols (OHCs) have demonstrated the ability to act as agonists or antagonists of the hedgehog (Hh) signaling pathway, a developmental signaling pathway that has been implicated as a potential therapeutic target in a variety of human diseases. These OHCs are known to modulate Hh signaling through direct binding interactions with the N-terminal cysteine rich domain (CRD) of Smoothened, a key regulator of Hh signal transduction. MethodsHomology modeling, molecular dynamics simulations, and MM/GBSA energy calculations were utilized to explore binding interactions between the OHC scaffold and the human Smoothened CRD. Follow-up cellular assays explored the in vitro activity of potential Hh pathway modulators. ResultsStructural features that govern key molecular interactions between the Smoothened CRD and the OHC scaffold were identified. Orientation of the iso-octyl side chain as well as the overall entropy of the OHC-CRD complex are important for determining activity against the Hh pathway. OHC 9, which was previously thought to be inactive because it was not an Hh agonist, was identified as an inhibitor of Hh signal transmission. ConclusionsCalculated MM/GBSA binding energies for OHCs in complex with the CRD of Smoothened correlate well with in vitro Hh modulatory activity. Compounds with high affinity stabilize Smoothened and are antagonists, whereas compounds with reduced affinity allow a conformational change in Smoothened that results in pathway activation. General significanceComputational modeling and molecular dynamics simulations can be used to predict whether a small molecule that binds the Smoothened CRD will be an agonist or antagonist of the pathway.

Violaxanthin de-epoxidase disulphides and their role in activity and thermal stability.

Violaxanthin de-epoxidase (VDE) catalyses the conversion of violaxanthin to zeaxanthin at the lumen side of the thylakoids during exposure to intense light. VDE consists of a cysteine-rich N-terminal domain, a lipocalin-like domain and a negatively charged C-terminal domain. That the cysteines are important for the activity of VDE is well known, but in what way is less understood. In this study, wild-type spinach VDE was expressed in E. coli as inclusion bodies, refolded and purified to give a highly active and homogenous preparation. The metal content (Fe, Cu, Ni, Mn, Co and Zn) was lower than 1 mol% excluding a metal-binding function of the cysteines. To investigate which of the 13 cysteines that could be important for the function of VDE, we constructed mutants where the cysteines were replaced by serines, one by one. For 12 out of 13 mutants the activity dropped by more than 99.9 %. A quantification of free cysteines showed that only the most N-terminal of these cysteines was in reduced form in the native VDE. A disulphide pattern in VDE of C9–C27, C14–C21, C33–C50, C37–C46, C65–C72 and C118–C284 was obtained after digestion of VDE with thermolysin followed by mass spectroscopy analysis of reduced versus non-reduced samples. The residual activity found for the mutants showed a variation that was consistent with the results obtained from mass spectroscopy. Reduction of the disulphides resulted in loss of a rigid structure and a decrease in thermal stability of 15 °C.

Dystrophin Genotype–Cardiac Phenotype Correlations in Duchenne and Becker Muscular Dystrophies Using Cardiac Magnetic Resonance Imaging

Duchenne and Becker muscular dystrophies are caused by mutations in dystrophin. Cardiac manifestations vary broadly, making prognosis difficult. Current dystrophin genotype-cardiac phenotype correlations are limited. For skeletal muscle, the reading-frame rule suggests in-frame mutations tend to yield milder phenotypes. We performed dystrophin genotype-cardiac phenotype correlations using a protein-effect model and cardiac magnetic resonance imaging. A translational model was applied to patient-specific deletion, indel, and nonsense mutations to predict exons and protein domains present within truncated dystrophin protein. Patients were dichotomized into predicted present and predicted absent groups for exons and protein domains of interest. Development of myocardial fibrosis (represented by late gadolinium enhancement [LGE]) and depressed left ventricular ejection fraction (LVEF) were compared. Patients (n = 274) with predicted present cysteine-rich domain (CRD), C-terminal domain (CTD), and both the N-terminal actin-binding and cysteine-rich domains (ABD1 + CRD) had a decreased risk of LGE and trended toward greater freedom from LGE. Patients with predicted present CTD (exactly the same as those with in-frame mutations) and ABD1 + CRD trended toward decreased risk of and greater freedom from depressed LVEF. In conclusion, genotypes previously implicated in altering the dystrophinopathic cardiac phenotype were not significantly related to LGE and depressed LVEF. Patients with predicted present CRD, CTD/in-frame mutations, and ABD1 + CRD trended toward milder cardiac phenotypes, suggesting that the reading-frame rule may be applicable to the cardiac phenotype. Genotype-phenotype correlations may help predict the cardiac phenotype for dystrophinopathic patients and guide future therapies.