N-terminal Segment Research Articles

Conserved GxxxG and WN motifs of MIC13 are essential for bridging two MICOS subcomplexes

Mitochondrial ultrastructure is highly adaptable and undergoes dynamic changes upon physiological and energetic cues. MICOS (mitochondrial contact site and cristae organizing system), a large oligomeric protein complex, maintains mitochondrial ultrastructure as it is required for formation of crista junctions (CJs) and contact sites. MIC13 acts as a critical bridge between two MICOS subcomplexes. Deletion of MIC13 causes loss of CJs resulting in cristae accumulating as concentric rings and specific destabilization of the MIC10-subcomplex. Mutations in MIC13 are associated with infantile lethal mitochondrial hepato-encephalopathy, yet functional regions within MIC13 were not known. To identify and characterize such regions, we systemically generated 20 amino-acids deletion variants across the length of MIC13. While deletion of many of these regions of MIC13 is dispensable for its stability, the N-terminal region and a stretch between amino acid residues 84 and 103 are necessary for the stability and functionality of MIC13. We could further locate conserved motifs within these regions and found that a GxxxG motif in the N-terminal transmembrane segment and an internal WN motif are essential for stability of MIC13, formation of the MIC10-subcomplex, interaction with MIC10- and MIC60-subcomplexes and maintenance of cristae morphology. The GxxxG motif is required for membrane insertion of MIC13. Overall, we systematically found important conserved residues of MIC13 that are required to perform the bridging between the two MICOS subcomplexes. The study improves our understanding of the basic molecular function of MIC13 and has implications for its role in the pathogenesis of a severe mitochondrial disease.

SNAP-tag-enabled super-resolution imaging reveals constitutive and agonist-dependent trafficking of GPR56 in pancreatic β-cells

ObjectiveMembers of the adhesion G protein-coupled receptor (aGPCR) subfamily are important actors in metabolic processes, with GPR56 (ADGRG1) emerging as a possible target for type 2 diabetes therapy. GPR56 can be activated by collagen III, its endogenous ligand, and by a synthetic seven amino-acid peptide (TYFAVLM; P7) contained within the GPR56 Stachel sequence. However, the mechanisms regulating GPR56 trafficking dynamics and agonist activities are not yet clear. MethodsHere, we introduced SNAPf-tag into the N-terminal segment of GPR56 to monitor GPR56 cellular activity in situ. Confocal and super-resolution microscopy were used to investigate the trafficking pattern of GPR56 in native MIN6 β-cells and in MIN6 β-cells where GPR56 had been deleted by CRISPR-Cas9 gene editing. Insulin secretion, changes in intracellular calcium, and β-cell apoptosis were determined by radioimmunoassay, single-cell calcium microfluorimetry, and measuring caspase 3/7 activities, respectively, in MIN6 β-cells and human islets. ResultsSNAP-tag labelling indicated that GPR56 predominantly underwent constitutive internalisation in the absence of an exogenous agonist, unlike GLP-1R. Collagen III further stimulated GPR56 internalisation, whereas P7 was without significant effect. The overexpression of GPR56 in MIN6 β-cells did not affect insulin secretion. However, it was associated with reduced β-cell apoptosis, while the deletion of GPR56 made MIN6 β-cells more susceptible to cytokine-induced apoptosis. P7 induced a rapid increase in the intracellular calcium in MIN6 β-cells (in a GPR56-dependent manner) and human islets, and it also caused a sustained and reversible increase in insulin secretion from human islets. Collagen III protected human islets from cytokine-induced apoptosis, while P7 was without significant effect. ConclusionsThese data indicate that GPR56 exhibits both agonist-dependent and -independent trafficking in β-cells and suggest that while GPR56 undergoes constitutive signalling, it can also respond to its ligands when required. We have also identified that constitutive and agonist-dependent GPR56 activation is coupled to protect β-cells against apoptosis, offering a potential therapeutic target to maintain β-cell mass in type 2 diabetes.

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.

A Good Way to Reduce Screening for Retinopathy of Prematurity: Development of the ROP Model in a China Preterm Population.

Importance: Retinopathy of prematurity (ROP) is a preventable cause of blindness in children. Without treatment, more than 45% of eyes may suffer permanent vision loss. Current ROP screening guidelines, which include a range of birth weights (BWs) and gestational ages (GAs), may require screening many low-risk preemies who might develop severe ROP.Method: All high-risk infants in the neonatal intensive care unit (NICU) of the First Affiliated Hospital of Zhengzhou University from 2017 to 2021 were included in this retrospective cohort study. Each of the 27 candidate risk factors was evaluated in univariate analysis and adjusted for known risk factors (i.e., GA and BW). The significant results were analyzed in a backward selection multivariate logistic regression model. Receiver operating characteristic (ROC) curves and a nomogram were drawn.Results: The study included 2,040 infants who underwent ROP screening. The weight gain rate [OR, 2.65; 95% confidence interval (CI), 1.49–1.21 ≤ 12 g/d vs. > 18 g/d; P = 0.001], blood transfusion (OR, 2.03; 95% CI, 1.14–3.64; P = 0.017), invasive mechanical ventilation (OR, 1.74; 95% CI, 1.15–2.66; P = 0.009) and N-terminal segment of pro-B-type natriuretic peptide (NT-proBNP) ≥ 25,000 ng/L (OR, 1.51; 95% CI, 1.00–2.28; P = 0.048) were four new statistically independent risk factors in addition to GA and BW. The area under the curve (AUC) of the final multivariate model was 0.90 (95% CI, 0.88–0.92; P < 0.001).Conclusions and Relevance: These findings add to our understanding of ROP screening because they include all eligible infants rather than only high-risk infants, as in previous studies. Under the control of BW and GA, low weight gain rate, increased number of blood transfusion, invasive mechanical ventilation and NT-proBNP ≥ 25,000 ng/L were “new” statistically independent risk factors for ROP. The ROP risk can be calculated manually or represented by a nomogram for clinical use.

Upregulation of Alternatively Glycosylated Smoothened (SMO) Is Involved in Doxorubicin (DXR) Resistance in Diffuse Large B-Cell Lymphoma

▪Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoid malignancy in adults. Chemoresistance remains the most urgent clinical challenge in the management of DLBCL patients. The salvage therapy options for patients with relapsed disease are poor (responses rates less than 20%) and survival is usually short. Therefore, the development of less toxic yet effective therapeutic approaches for DLBCL patients will address a significant immediate medical need. Smoothened (SMO), a Frizzled-class G-protein-coupled-receptor (GPCR), is activated by cholesterol and regulated indirectly by Hedgehog (Hh) ligands through the release of PTCH1-mediated suppression. SMO contains an N-terminal extracellular segment with the cysteine-rich domain (CRD), a heptahelical transmembrane domain (TMD), and a C-terminal intracellular segment. Although SMO localizes in cilia to function as Hh signal transducer, our data support that SMO is also a constituent of lipid rafts, and plays a fundamental role in lipid raft organization in DLBCL. SMO in doxorubicin (DXR) resistant cell lines shows increased glycosylation of its extracellular segments and drug resistance is sensitive to modulation of cellular cholesterol levels, both features associated with protein localization to lipid raft and lipid raft composition, a poorly understood aspect in drug resistance. To improve our current therapeutic approaches for lymphoma patients we need to increase our understanding of the cellular mechanisms that contribute to tumor progression and drug resistance.We established a DXR-resistant cell line (SUDHL4) through long-term exposure to escalating doses of DXR (DXR-resistant cell line: virtually no cell death up to a DXR concentration of 120 nM; parental cell line DXR-IC50: 40nM). We observed that DXR-resistant SUDHL4 cells were also resistant to escalating doses of vincristine in comparison with the parental (sensitive) cells. Studies on the response of DXR resistant lymphoma cells to DXR confirmed high levels of DNA damage in resistant cells (measured by phosphorylation of histone H2AX) confirming that DXR resistance was not due to increase drug efflux. In addition, in this experimental model, both parental and DXR-resistant lymphoma cells have mutated TP53 (missense mutation p.V157A) showing that TP53 deficiency was not a factor in inducing DXR resistance.Several lines of evidence support a direct role of SMO in the DXR-resistance observed in DLBCL cells. First, DXR-resistant lymphoma cells in comparison with the parental cells showed a strong increase in SMO mRNA, but not its canonical downstream target of signaling, the transcription factor GLI1. Concordantly, SMO protein levels were substantially elevated in DXR-resistant cells (as detected by immunofluorescence and western blot). Moreover, upregulation of the expression of SMO was detected by immunohistochemistry in 4 of 5 relapsed DLBCL tumors in comparison with the matched tumors at the time of the initial diagnosis and before chemotherapy treatment. Second, we found that SMO is localized in the lipid rafts and mediates the assembly of a TRAF6 E3 ligase-dependent machinery that enhances AKT signaling. Additionally, we observed that SMO is highly glycosylated in DXR-resistant SUDHL4 cells but not in the parental cell line; previously, it has been found that SMO has 4 potential N-glycosilation sites that are dispensable for canonical Hh signaling but needed to induce a non-canonical SMO signal through Gα1. Finally, depletion of SMO using shRNA and a lentiviral-tetracycline inducible system and pharmacologically inhibition of SMO using the classic SMO inhibitor (cyclopamine-KAAD) or a cholesterol synthesis inhibitor (lovastatin) re-established sensitivity to DXR in DXR-resistant cells. Interestingly, lovastatin did not modify DXR sensitivity in the parental cell line. In summary, this study suggests a novel function of SMO in the development of DXR-resistance in DLBCL and opens new opportunities to regain or enhance DXR sensitivity through targeting this novel mechanism. DisclosuresNo relevant conflicts of interest to declare.

3D architecture and structural flexibility revealed in the subfamily of large glutamate dehydrogenases by a mycobacterial enzyme

Glutamate dehydrogenases (GDHs) are widespread metabolic enzymes that play key roles in nitrogen homeostasis. Large glutamate dehydrogenases composed of 180 kDa subunits (L-GDHs180) contain long N- and C-terminal segments flanking the catalytic core. Despite the relevance of L-GDHs180 in bacterial physiology, the lack of structural data for these enzymes has limited the progress of functional studies. Here we show that the mycobacterial L-GDH180 (mL-GDH180) adopts a quaternary structure that is radically different from that of related low molecular weight enzymes. Intersubunit contacts in mL-GDH180 involve a C-terminal domain that we propose as a new fold and a flexible N-terminal segment comprising ACT-like and PAS-type domains that could act as metabolic sensors for allosteric regulation. These findings uncover unique aspects of the structure-function relationship in the subfamily of L-GDHs.

Broadly reactive human antibodies prevent and treat pneumococcal infection

Abstract Streptococcus pneumoniae remains a leading cause of bacterial pneumonia despite the widespread introduction of vaccines for disease prevention. While vaccines have been effective at reducing the incidence of most vaccine-included serotypes, a rise in infection due to non-vaccine serotypes, and moderate efficacy against some vaccine included serotypes have contributed to high disease incidence, particularly in the elderly. Additionally, numerous isolates of S. pneumoniae are antibiotic resistant or multi-drug resistant. Several highly conserved pneumococcal proteins that are prevalent in the majority of serotypes have been examined and tested as potential vaccines in preclinical and clinical trials. We isolated the first human monoclonal antibodies (mAbs) (PhtD3, PhtD6, PhtD7, PhtD8, PspA16) against the pneumococcal histidine triad protein (PhtD), and the pneumococcal surface protein A (PspA), two conserved and protective antigens. mAbs to PhtD target diverse epitopes spanning the entire PhtD protein, and mAb PspA16 targets the N-terminal segment of PspA. The PhtD-specific mAbs were found to bind to multiple serotypes, while PspA16 serotype breadth was limited. In addition, we examined the prophylactic and therapeutic efficacy of mAb PhtD3 in several mouse models of pneumococcal pneumonia and sepsis. mAb PhtD3 prolonged the survival of infected mice when administered 2 hours before infection or 24 hours after infection. Additionally, mAb PhtD3 was effective against pneumococcal serotypes 4 and 3, the latter of which is a leading cause of invasive pneumococcal disease. Overall, our results provide new therapeutic reagents for disease prevention, and identify regions on PhtD and PspA recognized by human B cells.

Rational Design of Peptide Inhibitor Against Amyloidogenesis-Correlated Membrane Disruption by Merozoite Surface Protein 2

New drugs and vaccines are urgently needed for malaria control owing to the increasing prevalence of drug-resistant Plasmodium strains. Malarial merozoite surface protein 2 (MSP2), potentially playing a role in parasite invasion into the host red blood cells, could be a target for anti-malaria drug development. MSP2 is suggested to cause membrane disruption that is correlated with amyloidogenesis of the protein, and thus peptides that can inhibit this property may be exploited as drug candidates against malaria. In this study, we designed peptides by introducing residues that favor β-turn formation into the amyloidogenic N-terminal segment between the 8th and 22nd residues of MSP2 (MSP28–22), and evaluated the ability of these derivative peptides to inhibit MSP2 amyloidogenesis and membrane disruption. One of the derivative peptides, M5, with a β-turn-prone sequence DPDG being inserted within MSP28–22, though did not form a rigid β-turn structure, did inhibit aggregation and membrane interaction by both itself and the wild-type peptide. Insertion of a β-turn-prone sequence may thus be a potential strategy to develop safe and effective peptide inhibitors against amyloidogeneic protein/peptide.

Atomic Resolution Map of Hierarchical Self-Assembly for an Amyloidogenic Protein Probed through Thermal 15N-R2 Correlation Matrices.

Soluble oligomers formed by amyloidogenic intrinsically disordered proteins are some of the most cytotoxic species linked to neurodegeneration. Due to the transient and heterogeneous nature of such oligomeric intermediates, the underlying self-association events often remain elusive. NMR relaxation measurements sensitive to zero-frequency spectral densities (J(0)), such as the 15N - R2 rates, are ideally suited to map sites of self-association at atomic resolution without the need of exogenous labels. Such experiments exploit the dynamic exchange between NMR visible monomers and slowly tumbling oligomers. However,15N - R2 rates are also sensitive to intrinsic monomer dynamics, and it is often difficult to discern these contributions from those arising from exchange with oligomers. Another challenge pertains to defining a hierarchy of self-association. Here, using the archetypical amyloidogenic protein alpha synuclein (αS), we show that the temperature-dependence of 15N - R2 effectively identifies self-association sites with reduced bias from internal dynamics. The key signature of the residues involved in self-association is a nonlinear temperature-dependence of 15N - R2 with a positive ΔR2/ΔT slope. These two hallmarks are systematically probed through a thermal R2 correlation matrix, from which the network of residues involved in self-association as well as the hierarchy of αS self-association sites is extracted through agglomerative clustering. We find that aggregation is initiated by residues within the NAC region that is solvent inaccessible in αS fibrils and eventually extends to the N-terminal segment harboring familial PD mutations. These hierarchical self-association maps help dissect the essential drivers of oligomerization and reveal how amyloid inhibitors affect oligomer formation.

Mutagenesis of a Lotus japonicus GSK3β/Shaggy-like kinase reveals functionally conserved regulatory residues

The glycogen synthase kinases 3 family (GSK3s/SKs; serine/threonine protein kinases) is conserved throughout eukaryotic evolution from yeast to plants and mammals. We studied a plant SK kinase from Lotus japonicus (LjSK1), previously implicated in nodule development, by enzyme kinetics and mutagenesis studies to compare it to mammalian homologues. Using a phosphorylated peptide as substrate, LjSK1 displays optimum kinase activity at pH 8.0 and 20 °C following Michaelis-Menten kinetics with Km and Vmax values of 48.2 μM and 111.6 nmol/min/mg, respectively, for ATP. Mutation of critical residues, as inferred by sequence comparison to the human homologue GSK3β and molecular modeling, showed a conserved role for Lys167, while residues conferring substrate specificity in the human enzyme are not as significant in modulating LjSK1 substrate specificity. Mutagenesis studies also indicate a regulation mechanism for LjSK1 via proteolysis since removal of a 98 residue long N-terminal segment increases its catalytic efficiency by almost two-fold. In addition, we evaluated the alteration of LjSK1 kinase activity in planta, by overexpressing the mutant variants in hairy-roots and a phenotype in nodulation and lateral root development was verified.

Oligomerized liprin-α promotes phase separation of ELKS for compartmentalization of presynaptic active zone proteins

Synaptic scaffold proteins (e.g., liprin-α, ELKS, RIM, and RIM-BP) orchestrate ion channels, receptors, and enzymes at presynaptic terminals to form active zones for neurotransmitter release. The underlying mechanism of the active zone assembly remains elusive. Here, we report that liprin-α proteins have the potential to oligomerize through the N-terminal coiled-coil region. Our structural and biochemical characterizations reveal that a gain-of-function mutation promotes the self-assembly of the coiled coils in liprin-α2 by disrupting intramolecular interactions and promoting intermolecular interactions. By enabling multivalent interactions with ELKS proteins, the oligomerized coiled-coil region of liprin-α2 enhances the phase separation of the ELKS N-terminal segment. We further show that liprin-α2, by regulating the interplay between two phase separations of ELKS and RIM/RIM-BP, controls the protein distributions. These results imply that the complicated protein-protein interactions allow liprin-α to function with the active zone scaffolds and compartmentalize protein assemblies to achieve comprehensive functions in the active zone.

PH-dependent and dynamic interactions of cystatin C with heparan sulfate

Cystatin C (Cst-3) is a potent inhibitor of cysteine proteases with diverse biological functions. As a secreted protein, the potential interaction between Cst-3 and extracellular matrix components has not been well studied. Here we investigated the interaction between Cst-3 and heparan sulfate (HS), a major component of extracellular matrix. We discovered that Cst-3 is a HS-binding protein only at acidic pH. By NMR and site-directed mutagenesis, we identified two HS binding regions in Cst-3: the highly dynamic N-terminal segment and a flexible region located between residue 70-94. The composition of the HS-binding site by two highly dynamic halves is unique in known HS-binding proteins. We further discovered that HS-binding severely impairs the inhibitory activity of Cst-3 towards papain, suggesting the interaction could actively regulate Cst-3 activity. Using murine bone tissues, we showed that Cst-3 interacts with bone matrix HS at low pH, again highlighting the physiological relevance of our discovery.

The intrinsically disordered N-terminus of the voltage-dependent anion channel.

The voltage-dependent anion channel (VDAC) is a critical β-barrel membrane protein of the mitochondrial outer membrane, which regulates the transport of ions and ATP between mitochondria and the cytoplasm. In addition, VDAC plays a central role in the control of apoptosis and is therefore of great interest in both cancer and neurodegenerative diseases. Although not fully understood, it is presumed that the gating mechanism of VDAC is governed by its N-terminal region which, in the open state of the channel, exhibits an α-helical structure positioned midway inside the pore and strongly interacting with the β-barrel wall. In the present work, we performed molecular simulations with a recently developed force field for disordered systems to shed new light on known experimental results, showing that the N-terminus of VDAC is an intrinsically disordered region (IDR). First, simulation of the N-terminal segment as a free peptide highlighted its disordered nature and the importance of using an IDR-specific force field to properly sample its conformational landscape. Secondly, accelerated dynamics simulation of a double cysteine VDAC mutant under applied voltage revealed metastable low conducting states of the channel representative of closed states observed experimentally. Related structures were characterized by partial unfolding and rearrangement of the N-terminal tail, that led to steric hindrance of the pore. Our results indicate that the disordered properties of the N-terminus are crucial to properly account for the gating mechanism of VDAC.

Genetically Engineered Addition of a Ca2+ Buffering Capacity in Mouse Cardiac Muscle Thin Filament using a Glu-Rich Segment of Insect Troponin T Alters Cardiac Performance

In addition to a core function in Ca2+ regulation of muscle contraction, insect troponin T (TnT) has a unique glutamic acid-rich long C-terminal extension. With sequence similarity to this diverged structure, the N-terminal segment of avian flight muscle TnT binds Ca2+ with a physiologically relevant affinity (Zhang et al., Biochemistry 43:2645-55, 2004). Deletion of the C-terminal extension of Drosophila TnT significantly decreased muscle and heart functions (Cao et al., JBC 295:3794-3807, 2020). We propose a hypothesis that this structure serves as a myofilament Ca2+ reservoir to reduce the amount of Ca2+ cycled by Ca2+ pumps during muscle contraction and relaxation. A CRISP/Cas9 knock-in (KI) mouse model was constructed to replace the N-terminal variable region of cardiac TnT with 51 Glu residues derived from the C-terminal extension of bee TnT. Cardiac function is studied using ex vivo working hearts and tested for responses to beta-adrenergic stimulation or L-Ca2+ channel agonist Bay-K8644. At baseline, cTnT-51E hearts showed lower systolic velocity than that of WT control. Upon isoproterenol or Bay-K8644 treatment, cTnT-51E and wild type hearts both had positive responses in performance. However, cTnT-51E hearts exhibited significantly more increases in ventricular peak systolic pressure and diastolic velocity but still lower stroke volume as compared to that of WT controls. This phenotype is likely due to an altered response to the increase in intracellular calcium. Further analysis of Ca2+ responses of skinned muscle sections, cardiac energetic efficiency and intracellular Ca2+ transient are underway to establish the novel Ca2+ buffering capacity of TnT and its function in contractile kinetics of striated muscle.

Structural Interplays in the Flexible N-Terminus and Scaffolding Domain of Human Membrane Protein Caveolin 3.

Caveolins are critical for the formation of caveolae, which are small invaginations of the plasma membrane involved in a variety of biological processes. Caveolin 3 (Cav3), one of three caveolin isoforms, is an integral membrane protein mainly expressed in muscle tissues. Although various human diseases associated with Cav3 have been reported, structural characterization of Cav3 in the membrane has not been investigated in enough depth to understand the structure–function relationship. Here, using solution NMR, we characterized membrane association, structural communications, and molecular dynamics of the monomeric Cav3 in detergent micelle environment, particularly focused on the whole N-terminal part that is composed of the flexible N-terminus and the scaffolding domain. The results revealed a complicated structural interplay of the individual segments composing the whole N-terminal part, including the pH-dependent helical region, signature motif-like region, signature motif, and scaffolding domain. Collectively, the present study provides novel structural insights into the whole N-terminal part of Cav3 that plays important biological roles in cellular processes and diseases. In particular, given that several disease-related mutations are located at the whole N-terminal part of Cav3, the sophisticated communications in the whole N-terminal segments are likely to have relevance to the molecular basis of Cav3-related disease.

The structure of the cysteine-rich region from human histone-lysine N-methyltransferase EHMT2 (G9a)

Euchromatic histone-lysine N-methyltransferase 1 (EHMT1; G9a-like protein; GLP) and euchromatic histone-lysine N-methyltransferase 2 (EHMT2; G9a) are protein lysine methyltransferases that regulate gene expression and are essential for development and the ability of organisms to change and adapt. In addition to ankyrin repeats and the catalytic SET domain, the EHMT proteins contain a unique cysteine-rich region (CRR) that mediates protein–protein interactions and recruitment of the methyltransferases to specific sites in chromatin. We have determined the structure of the CRR from human EHMT2 by X-ray crystallography and show that the CRR adopts an unusual compact fold with four bound zinc atoms. The structure consists of a RING domain preceded by a smaller zinc-binding motif and an N-terminal segment. The smaller zinc-binding motif straddles the N-terminal end of the RING domain, and the N-terminal segment runs in an extended conformation along one side of the structure and interacts with both the smaller zinc-binding motif and the RING domain. The interface between the N-terminal segment and the RING domain includes one of the zinc atoms. The RING domain is partially sequestered within the CRR and unlikely to function as a ubiquitin ligase.

A straightforward methodology to overcome solubility challenges for N-terminal cysteinyl peptide segments used in native chemical ligation.

One of the main limitations encountered during the chemical synthesis of proteins through native chemical ligation (NCL) is the limited solubility of some of the peptide segments. The most commonly used solution to overcome this problem is to derivatize the segment with a temporary solubilizing tag. Conveniently, the tag can be introduced on the thioester segment in such a way that it is removed concomitantly with the NCL reaction. We herein describe a generalization of this approach to N-terminal cysteinyl segment counterparts, using a straightforward synthetic approach that can be easily automated from commercially available building blocks, and applied it to a well-known problematic target, SUMO-2.

Moieties of Complement iC3b Recognized by the I-domain of Integrin αXβ2.

Complement fragment iC3b serves as a major opsonin for facilitating phagocytosis via its interaction with complement receptors CR3 and CR4, also known by their leukocyte integrin family names, αMβ2 and αXβ2, respectively. Although there is general agreement that iC3b binds to the αM and αX I-domains of the respective β2-integrins, much less is known regarding the regions of iC3b contributing to the αX I-domain binding. In this study, using recombinant αX I-domain, as well as recombinant fragments of iC3b as candidate binding partners, we have identified two distinct binding moieties of iC3b for the αX I-domain. They are the C3 convertase-generated N-terminal segment of the C3b α’-chain (α’NT) and the factor I cleavage-generated N-terminal segment in the CUBf region of α-chain. Additionally, we have found that the CUBf segment is a novel binding moiety of iC3b for the αM I-domain. The CUBf segment shows about a 2-fold higher binding activity than the α’NT for αX I-domain. We also have shown the involvement of crucial acidic residues on the iC3b side of the interface and basic residues on the I-domain side.

Cryo-electron Microscopy Structure, Assembly, and Mechanics Show Morphogenesis and Evolution of Human Picobirnavirus.

Despite their diversity, most double-stranded-RNA (dsRNA) viruses share a specialized T=1 capsid built from dimers of a single protein that provides a platform for genome transcription and replication. This ubiquitous capsid remains structurally undisturbed throughout the viral cycle, isolating the genome to avoid triggering host defense mechanisms. Human picobirnavirus (hPBV) is a dsRNA virus frequently associated with gastroenteritis, although its pathogenicity is yet undefined. Here, we report the cryo-electron microscopy (cryo-EM) structure of hPBV at 2.6-Å resolution. The capsid protein (CP) is arranged in a single-shelled, ∼380-Å-diameter T=1 capsid with a rough outer surface similar to that of dsRNA mycoviruses. The hPBV capsid is built of 60 quasisymmetric CP dimers (A and B) stabilized by domain swapping, and only the CP-A N-terminal basic region interacts with the packaged nucleic acids. hPBV CP has an α-helical domain with a fold similar to that of fungal partitivirus CP, with many domain insertions in its C-terminal half. In contrast to dsRNA mycoviruses, hPBV has an extracellular life cycle phase like complex reoviruses, which indicates that its own CP probably participates in cell entry. Using an in vitro reversible assembly/disassembly system of hPBV, we isolated tetramers as possible assembly intermediates. We used atomic force microscopy to characterize the biophysical properties of hPBV capsids with different cargos (host nucleic acids or proteins) and found that the CP N-terminal segment not only is involved in nucleic acid interaction/packaging but also modulates the mechanical behavior of the capsid in conjunction with the cargo.IMPORTANCE Despite intensive study, human virus sampling is still sparse, especially for viruses that cause mild or asymptomatic disease. Human picobirnavirus (hPBV) is a double-stranded-RNA virus, broadly dispersed in the human population, but its pathogenicity is uncertain. Here, we report the hPBV structure derived from cryo-electron microscopy (cryo-EM) and reconstruction methods using three capsid protein variants (of different lengths and N-terminal amino acid compositions) that assemble as virus-like particles with distinct properties. The hPBV near-atomic structure reveals a quasisymmetric dimer as the structural subunit and tetramers as possible assembly intermediates that coassemble with nucleic acids. Our structural studies and atomic force microscopy analyses indicate that hPBV capsids are potentially excellent nanocages for gene therapy and targeted drug delivery in humans.

Application of 2,2′‐dipyridyl disulfide‐mediated thiazolidine ring‐opening reaction to glycoprotein synthesis: Total chemical synthesis of evasin‐3

Thiazolidine ring-opening reaction is one of the key steps in protein chemical synthesis via sequential native chemical ligation strategy. We recently developed a novel thiazolidine ring-opening reaction with 2,2'-dipyridyl disulfide (DPDS). In order to investigate the applicability of this reaction to glycoprotein synthesis, we synthesized evasin-3, a cysteine-rich glycoprotein with chemokine-binding ability originally found in tick saliva. The sequence of evasin-3 was divided into three segments, and these segments were separately synthesized with the ordinary solid-phase peptide synthesis method. After the first ligation of middle and C-terminal segments, thiazolidine used as a protecting group of Cys residue at the N-terminus of the middle segment was converted to Cys with DPDS. In this thiazolidine ring-opening reaction, DPDS treatment did not affect the N-linked glycan moiety. After the second ligation with the N-terminal segment and the refolding reaction, evasin-3 could be obtained in good yield. The synthetic evasin-3 showed the binding ability specifically to CXCL chemokines. These results clearly indicate that this DPDS method is useful for glycoprotein synthesis.