NMR solution structure of an asymmetric intermolecular leaped V-shape G-quadruplex: selective recognition of the d(G2NG3NG4) sequence motif by a short linear G-rich DNA probe.

Aside from classical loops among G-quadruplexes, the unique leaped V-shape scaffold spans over three G-tetrads, without any intervening residues. This scaffold enables a sharp reversal of two adjacent strand directions and simultaneously participates in forming the G-tetrad core. These features make this scaffold itself distinctive and thus an essentially more accessible target. As an alternative to the conventional antisense method using a complementary chain, forming an intermolecular G-quadruplex from two different oligomers, in which the longer one as the target is captured by a short G-rich fragment, could be helpful for recognizing G-rich sequences and structural motifs. However, such an intermolecular leaped V-shape G-quadruplex consisting of DNA oligomers of quite different lengths has not been evaluated. Here, we present the first nuclear magnetic resonance (NMR) study of an asymmetric intermolecular leaped V-shape G-quadruplex assembled between an Oxytricha nova telomeric sequence d(G2T4G4T4G4) and a single G-tract fragment d(TG4A). Furthermore, we explored the selectivity of this short fragment as a potential probe, examined the kinetic discrimination for probing a specific mutant, and proposed the key sequence motif d(G2NG3NG4) essential for building the leaped V-shape G-quadruplexes.

Ca2+ regulates T-cell receptor activation by modulating the charge property of lipids.

Ionic protein-lipid interactions are critical for the structure and function of membrane receptors, ion channels, integrins and many other proteins. However, the regulatory mechanism of these interactions is largely unknown. Here we show that Ca(2+) can bind directly to anionic phospholipids and thus modulate membrane protein function. The activation of T-cell antigen receptor-CD3 complex (TCR), a key membrane receptor for adaptive immunity, is regulated by ionic interactions between positively charged CD3ε/ζ cytoplasmic domains (CD3(CD)) and negatively charged phospholipids in the plasma membrane. Crucial tyrosines are buried in the membrane and are largely protected from phosphorylation in resting T cells. It is not clear how CD3(CD) dissociates from the membrane in antigen-stimulated T cells. The antigen engagement of even a single TCR triggers a Ca(2+) influx and TCR-proximal Ca(2+) concentration is higher than the average cytosolic Ca(2+) concentration. Our biochemical, live-cell fluorescence resonance energy transfer and NMR experiments showed that an increase in Ca(2+) concentration induced the dissociation of CD3(CD) from the membrane and the solvent exposure of tyrosine residues. As a consequence, CD3 tyrosine phosphorylation was significantly enhanced by Ca(2+) influx. Moreover, when compared with wild-type cells, Ca(2+) channel-deficient T cells had substantially lower levels of CD3 phosphorylation after stimulation. The effect of Ca(2+) on facilitating CD3 phosphorylation is primarily due to the charge of this ion, as demonstrated by the fact that replacing Ca(2+) with the non-physiological ion Sr(2+) resulted in the same feedback effect. Finally, (31)P NMR spectroscopy showed that Ca(2+) bound to the phosphate group in anionic phospholipids at physiological concentrations, thus neutralizing the negative charge of phospholipids. Rather than initiating CD3 phosphorylation, this regulatory pathway of Ca(2+) has a positive feedback effect on amplifying and sustaining CD3 phosphorylation and should enhance T-cell sensitivity to foreign antigens. Our study thus provides a new regulatory mechanism of Ca(2+) to T-cell activation involving direct lipid manipulation.

Recombinant expression, purification and characterization of antimicrobial peptide ORBK in Escherichia coli.

ORBK (LKGCWTKSIPPKPCFK) is a cyclic cationic peptide that has potent antimicrobial properties and trypsin inhibitory activities. To explore a new approach for expressing ORBK in Escherichia coli, a sequence encoding ORBK was cloned into pET28a vector in which maltose-binding protein (MBP) was used as a fusion partner and an N-terminal 6-His as an affinity tag. Protein expression was induced with 0.5mM Isopropyl-thio-galactoside (IPTG) for 4h at 37°C. The recombinant ORBK was then purified by Ni affinity column and further digested with tobacco etch virus (TEV) enzyme. The cleaved ORBK peptide was separated from MBP fusion partner by reverse phase high performance liquid chromatography (RP-HPLC) and oxidized to obtain the cyclic form. Mass spectroscopy and nuclear magnetic resonance (NMR) spectroscopy were performed for ORBK characterization. Herein we have developed an effective and reliable method to express and purify ORBK which sets a solid foundation for future structural and functional studies.

Structural basis for the dynamic chaperoning of disordered clients by Hsp90.

Molecular chaperone heat shock protein 90 (Hsp90) is a ubiquitous regulator that fine-tunes and remodels diverse client proteins, exerting profound effects on normal biology and diseases. Unraveling the mechanistic details of Hsp90's function requires atomic-level insights into its client interactions throughout the adenosine triphosphate-coupled functional cycle. However, the structural details of the initial encounter complex in the chaperone cycle, wherein Hsp90 adopts an open conformation while engaging with the client, remain elusive. Here, using nuclear magnetic resonance spectroscopy, we determined the solution structure of Hsp90 in its open state, bound to a disordered client. Our findings reveal that Hsp90 uses two distinct binding sites, collaborating synergistically to capture discrete hydrophobic segments within client proteins. This bipartite interaction generates a versatile complex that facilitates rapid conformational sampling. Moreover, our investigations spanning various clients and Hsp90 orthologs demonstrate a pervasive mechanism used by Hsp90 orthologs to accommodate the vast array of client proteins. Collectively, our work contributes to establish a unified conceptual and mechanistic framework, elucidating the intricate interplay between Hsp90 and its clients.

A comprehensive whole genome database of ethnic minority populations.

China, is characterized by its remarkable ethnical diversity, which necessitates whole genome variation data from multiple populations as crucial tools for advancing population genetics and precision medical research. However, there has been a scarcity of research concentrating on the whole genome of ethnic minority groups. To fill this gap, we developed the Guizhou Multi-ethnic Genome Database (GMGD). It comprises whole genome sequencing data from 476 healthy unrelated individuals spanning 11 ethnic minorities groups in Guizhou Province, Southwest China, including Bouyei, Dong, Miao, Yi, Bai, Gelo, Zhuang, Tujia, Yao, Hui, and Sui. The GMGD database comprises more than 16.33 million variants in GRCh38 and 16.20 million variants in GRCh37. Among these, approximately 11.9% (1,956,322) of the variants in GRCh38 and 18.5% (3,009,431) of the variants in GRCh37 are entirely new and do not exist in the dbSNP database. These novel variants shed light on the genetic diversity landscape across these populations, providing valuable insights with an average coverage of 5.5 ×. This makes GMGD the largest genome-wide database encompassing the most diverse ethnic groups to date. The GMGD interactive interface facilitates researchers with multi-dimensional mutation search methods and displays population frequency differences among global populations. Furthermore, GMGD is equipped with a genotype-imputation function, enabling enhanced capabilities for low-depth genomic research or targeted region capture studies. GMGD offers unique insights into the genomic variation landscape of different ethnic groups, which are freely accessible at https://db.cngb.org/pop/gmgd/ .

SNX16 Regulates the Recycling of E-Cadherin through a Unique Mechanism of Coordinated Membrane and Cargo Binding.

E-Cadherin is a major component of adherens junctions on cell surfaces. SNX16 is a unique member of sorting nexins that contains a coiled-coil (CC) domain downstream of the PX domain. We report here that SNX16 regulates the recycling trafficking of E-cadherin. We solved the crystal structure of PX-CC unit of SNX16 and revealed a unique shear shaped homodimer. We identified a novel PI3P binding pocket in SNX16 that consists of both the PX and the CC domains. Surprisingly, we showed that the PPII/α2 loop, which is generally regarded as a membrane insertion loop in PX family proteins, is involved in the E-cadherin binding with SNX16. We then proposed a multivalent membrane binding model for SNX16. Our study postulates a new mechanism for coordinated membrane binding and cargo binding for SNX family proteins in general, and provide novel insights into recycling trafficking of E-cadherin.

Insights into the molecular recognition of the granuphilin C2A domain with PI(4,5)P2.

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a key player in regulating the process of excytosis, including insulin secretion. Granuphilin, a tandem C2 domain containing protein, mediates the docking of insulin granules onto plasma membrane. The C2A domain plays key roles in this process through interaction with PI(4,5)P2. In this study, we have investigated the molecular recognition mechanism of granuphilin-C2A domain to PI(4,5)P2 head group, and further to PI(4,5)P2-nanodisc by NMR, ITC, MST and SEC methods. Our results demonstrate that PI(4,5)P2 binds to the concave surface of granuphilin-C2A domain. The key residues involved in the binding were validated by mutation analysis.