Structural Analysis of the Complex between Penta-EF-Hand ALG-2 Protein and Sec31A Peptide Reveals a Novel Target Recognition Mechanism of ALG-2

ALG-2, a 22-kDa penta-EF-hand protein, is involved in cell death, signal transduction, membrane trafficking, etc., by interacting with various proteins in mammalian cells in a Ca²⁺-dependent manner. Most known ALG-2-interacting proteins contain proline-rich regions in which either PPYPXnYP (type 1 motif) or PXPGF (type 2 motif) is commonly found. Previous X-ray crystal structural analysis of the complex between ALG-2 and an ALIX peptide revealed that the peptide binds to the two hydrophobic pockets. In the present study, we resolved the crystal structure of the complex between ALG-2 and a peptide of Sec31A (outer shell component of coat complex II, COPII; containing the type 2 motif) and found that the peptide binds to the third hydrophobic pocket (Pocket 3). While amino acid substitution of Phe⁸⁵, a Pocket 3 residue, with Ala abrogated the interaction with Sec31A, it did not affect the interaction with ALIX. On the other hand, amino acid substitution of Tyr¹⁸⁰, a Pocket 1 residue, with Ala caused loss of binding to ALIX, but maintained binding to Sec31A. We conclude that ALG-2 recognizes two types of motifs at different hydrophobic surfaces. Furthermore, based on the results of serial mutational analysis of the ALG-2-binding sites in Sec31A, the type 2 motif was newly defined.     

Structural and Functional Characterization of OXA-48: Insight into Mechanism and Structural Basis of Substrate Recognition and Specificity

Class D β-lactamase OXA-48 is widely distributed among Gram-negative bacteria and is an important determinant of resistance to the last-resort carbapenems. Nevertheless, the detailed mechanism by which this β-lactamase hydrolyzes its substrates remains poorly understood. In this study, the complex structures of OXA-48 and various β-lactams were modeled and the potential active site residues that may interact with various β-lactams were identified and characterized to elucidate their roles in OXA-48 substrate recognition. Four residues, namely S⁷⁰, K⁷³, S¹¹⁸, and K²⁰⁸ were found to be essential for OXA-48 to undergo catalytic hydrolysis of various penicillins and carbapenems both in vivo and in vitro. T²⁰⁹ was found to be important for hydrolysis of imipenem, whereas R²⁵⁰ played a major role in hydrolyzing ampicillin, imipenem, and meropenem most likely by forming a H-bond or salt-bridge between the side chain of these two residues and the carboxylate oxygen ions of the substrates. Analysis of the effect of substitution of alanine in two residues, W¹⁰⁵ and L¹⁵⁸, revealed their roles in mediating the activity of OXA-48. Our data show that these residues most likely undergo hydrophobic interaction with the R groups and the core structure of the β-lactam ring in penicillins and the carbapenems, respectively. Unlike OXA-58, mass spectrometry suggested a loss of the C6-hydroxyethyl group during hydrolysis of meropenem by OXA-48, which has never been demonstrated in Class D carbapenemases. Findings in this study provide comprehensive knowledge of the mechanism of the substrate recognition and catalysis of OXA-type β-lactamases.     

Structural Biology for the Molecular Insight between Aptamers and Target Proteins

Aptamers are promising therapeutic and diagnostic agents for various diseases due to their high affinity and specificity against target proteins. Structural determination in combination with multiple biochemical and biophysical methods could help to explore the interacting mechanism between aptamers and their targets. Regrettably, structural studies for aptamer–target interactions are still the bottleneck in this field, which are facing various difficulties. In this review, we first reviewed the methods for resolving structures of aptamer–protein complexes and for analyzing the interactions between aptamers and target proteins. We summarized the general features of the interacting nucleotides and residues involved in the interactions between aptamers and proteins. Challenges and perspectives in current methodologies were discussed. Approaches for determining the binding affinity between aptamers and target proteins as well as modification strategies for stabilizing the binding affinity of aptamers to target proteins were also reviewed. The review could help to understand how aptamers interact with their targets and how alterations such as chemical modifications in the structures affect the affinity and function of aptamers, which could facilitate the optimization and translation of aptamers-based theranostics.     

Structural Basis for the Function of the C-Terminal Proton Release Pathway in the Calcium Pump

The calcium pump (sarco/endoplasmic reticulum Ca²⁺-ATPase, SERCA) plays a major role in calcium homeostasis in muscle cells by clearing cytosolic Ca²⁺ during muscle relaxation. Active Ca²⁺ transport by SERCA involves the structural transition from a low-Ca²⁺ affinity E2 state toward a high-Ca²⁺ affinity E1 state of the pump. This structural transition is accompanied by the countertransport of protons to stabilize the negative charge and maintain the structural integrity of the transport sites and partially compensate for the positive charges of the two Ca²⁺ ions passing through the membrane. X-ray crystallography studies have suggested that a hydrated pore located at the C-terminal domain of SERCA serves as a conduit for proton countertransport, but the existence and function of this pathway have not yet been fully characterized. We used atomistic simulations to demonstrate that in the protonated E2 state and the absence of initially bound water molecules, the C-terminal pore becomes hydrated in the nanosecond timescale. Hydration of the C-terminal pore is accompanied by the formation of water wires that connect the transport sites with the cytosol. Water wires are known as ubiquitous proton-transport devices in biological systems, thus supporting the notion that the C-terminal domain serves as a conduit for proton release. Additional simulations showed that the release of a single proton from the transport sites induces bending of transmembrane helix M5 and the interaction between residues Arg762 and Ser915. These structural changes create a physical barrier against full hydration of the pore and prevent the formation of hydrogen-bonded water wires once proton transport has occurred through this pore. Together, these findings support the notion that the C-terminal proton release pathway is a functional element of SERCA and also provide a mechanistic model for its operation in the catalytic cycle of the pump.     

表面化学改性碳酸钙晶须对环氧树脂杂化材料的结构性能影响

利用硅烷偶联剂对碳酸钙晶须表面进行化学改性,研究了表面改性对环氧树脂/碳酸钙晶须复合材料的结构与性能的影响。研究结果表明,表面化学改性能够增加环氧树脂与碳酸钙晶须的相容性,增强碳酸钙晶须与环氧树脂之间的相互作用,改善碳酸钙晶须在环氧树脂中的分散。碳酸钙晶须与环氧树脂之间的界面相互作用是复合材料能够大幅度提高冲击强度的主要原因。     

A Trend to the Production of Calcium Hydroxide and Precipitated Calcium Carbonate with Defined Properties

In this paper, production of precipitated calcium carbonate (PCC) with required particle size and morphological structure, along with its dependence on technological parameters and the properties of Ca(OH)₂, is discussed. The effect of the reaction environment on the kinetics of CaO hydration and the formation of crystals in water suspension was established. A remarkable difference in the system's restoration ability after stirring was observed. The hydration process is initially controlled by a kinetic mechanism, followed by a diffusion‐controlled process. The dissolution speed of lime hydrated to suspension is eight times higher than that of lime hydrated to powder. Particles of hydrated lime appeared in various forms.     

硬脂酸钙对大豆分离蛋白塑料结构和性能的影响

将硬脂酸钙（CaSt2）与大豆分离蛋白（SPI）以不同比例混合,丙三醇作为增塑剂,经过涂膜的方式制备了改性SPI薄膜。通过衰减全反射-傅里叶变换红外光谱、X射线衍射、热分析、力学性能测试和水蒸气透过率系数等表征手段,研究了CaSt2改性SPI薄膜的结构和性能。结果表明,CaSt2在SPI中以两种结构形式存在,其中离子形式为主。与纯SPI薄膜相比,改性SPI薄膜的结晶度提高了70.4 %,内部结构更加致密。热分析表明,改性SPI薄膜的热稳定性增强。随着CaSt2含量的增加,改性SPI薄膜的水蒸气透过系数下降,当其含量为12 %时,水蒸气阻透能力提高了40 %;同时,断裂伸长率提高了67.63 %。     

Substrate Recognition Properties from an Intermediate Structural State of the UreA Transporter

Through a combination of comparative modeling, site-directed and classical random mutagenesis approaches, we previously identified critical residues for binding, recognition, and translocation of urea, and its inhibition by 2-thiourea and acetamide in the Aspergillus nidulans urea transporter, UreA. To deepen the structural characterization of UreA, we employed the artificial intelligence (AI) based AlphaFold2 (AF2) program. In this analysis, the resulting AF2 models lacked inward- and outward-facing cavities, suggesting a structural intermediate state of UreA. Moreover, the orientation of the W82, W84, N279, and T282 side chains showed a large variability, which in the case of W82 and W84, may operate as a gating mechanism in the ligand pathway. To test this hypothesis non-conservative and conservative substitutions of these amino acids were introduced, and binding and transport assessed for urea and its toxic analogue 2-thiourea, as well as binding of the structural analogue acetamide. As a result, residues W82, W84, N279, and T282 were implicated in substrate identification, selection, and translocation. Using molecular docking with Autodock Vina with flexible side chains, we corroborated the AF2 theoretical intermediate model, showing a remarkable correlation between docking scores and experimental affinities determined in wild-type and UreA mutants. The combination of AI-based modeling with classical docking, validated by comprehensive mutational analysis at the binding region, would suggest an unforeseen option to determine structural level details on a challenging family of proteins.     

Patient-Specific iPSCs-Based Models of Neurodegenerative Diseases: Focus on Aberrant Calcium Signaling

The development of cell reprogramming technologies became a breakthrough in the creation of new models of human diseases, including neurodegenerative pathologies. The iPSCs-based models allow for the studying of both hereditary and sporadic cases of pathologies and produce deep insight into the molecular mechanisms underlying neurodegeneration. The use of the cells most vulnerable to a particular pathology makes it possible to identify specific pathological mechanisms and greatly facilitates the task of selecting the most effective drugs. To date, a large number of studies on patient-specific models of neurodegenerative diseases has been accumulated. In this review, we focused on the alterations of such a ubiquitous and important intracellular regulatory pathway as calcium signaling. Here, we reviewed and analyzed the data obtained from iPSCs-based models of different neurodegenerative disorders that demonstrated aberrant calcium signaling.     

Ion Channel Partnerships: Odd and Not-So-Odd Couples Controlling Neuronal Ion Channel Function

The concerted function of the large number of ion channels expressed in excitable cells, including brain neurons, shapes diverse signaling events by controlling the electrical properties of membranes. It has long been recognized that specific groups of ion channels are functionally coupled in mediating ionic fluxes that impact membrane potential, and that these changes in membrane potential impact ion channel gating. Recent studies have identified distinct sets of ion channels that can also physically and functionally associate to regulate the function of either ion channel partner beyond that afforded by changes in membrane potential alone. Here, we review canonical examples of such ion channel partnerships, in which a Ca²⁺ channel is partnered with a Ca²⁺-activated K⁺ channel to provide a dedicated route for efficient coupling of Ca²⁺ influx to K⁺ channel activation. We also highlight examples of non-canonical ion channel partnerships between Ca²⁺ channels and voltage-gated K⁺ channels that are not intrinsically Ca²⁺ sensitive, but whose partnership nonetheless yields enhanced regulation of one or the other ion channel partner. We also discuss how these ion channel partnerships can be shaped by the subcellular compartments in which they are found and provide perspectives on how recent advances in techniques to identify proteins in close proximity to one another in native cells may lead to an expanded knowledge of other ion channel partnerships.     

PGRS Domain of Rv0297 of Mycobacterium tuberculosis Functions in A Calcium Dependent Manner

Mycobacterium tuberculosis (M.tb), the pathogen causing tuberculosis, is a major threat to human health worldwide. Nearly 10% of M.tb genome encodes for a unique family of PE/PPE/PGRS proteins present exclusively in the genus Mycobacterium. The functions of most of these proteins are yet unexplored. The PGRS domains of these proteins have been hypothesized to consist of Ca²⁺ binding motifs that help these intrinsically disordered proteins to modulate the host cellular responses. Ca²⁺ is an important secondary messenger that is involved in the pathogenesis of tuberculosis in diverse ways. This study presents the calcium-dependent function of the PGRS domain of Rv0297 (PE_PGRS5) in M.tb virulence and pathogenesis. Tandem repeat search revealed the presence of repetitive Ca²⁺ binding motifs in the PGRS domain of the Rv0297 protein (Rv0297PGRS). Molecular Dynamics simulations and fluorescence spectroscopy revealed Ca²⁺ dependent stabilization of the Rv0297PGRS protein. Calcium stabilized Rv0297PGRS enhances the interaction of Rv0297PGRS with surface localized Toll like receptor 4 (TLR4) of macrophages. The Ca²⁺ stabilized binding of Rv0297PGRS with the surface receptor of macrophages enhances its downstream consequences in terms of Nitric Oxide (NO) production and cytokine release. Thus, this study points to hitherto unidentified roles of calcium-modulated PE_PGRS proteins in the virulence of M.tb. Understanding the pathogenic potential of Ca²⁺ dependent PE_PGRS proteins can aid in targeting these proteins for therapeutic interventions.     

Cyclic Dipeptide: A Privileged Molecular Scaffold to Derive Structural Diversity and Functional Utility

Cyclic dipeptides (CDPs) are the simplest form of cyclic peptides with a wide range of applications from therapeutics to biomaterials. CDP is a versatile molecular platform endowed with unique properties such as conformational rigidity, intermolecular interactions, structural diversification through chemical synthesis, bioavailability and biocompatibility. A variety of natural products with the CDP core exhibit anticancer, antifungal, antibacterial, and antiviral activities. The inherent bioactivities have inspired the development of synthetic analogues as drug candidates and drug delivery systems. CDP plays a crucial role as conformation and molecular assembly directing core in the design of molecular receptors, peptidomimetics and fabrication of functional material architectures. In recent years, CDP has rapidly become a privileged scaffold for the design of advanced drug candidates, drug delivery agents, bioimaging, and biomaterials to mitigate numerous disease conditions. This review describes the structural diversification and multifarious biomedical applications of the CDP scaffold, discusses challenges, and provides future directions for the emerging field.     

The Expression of Non-Coding RNAs and Their Target Molecules in Rheumatoid Arthritis: A Molecular Basis for Rheumatoid Pathogenesis and Its Potential Clinical Applications

Rheumatoid arthritis (RA) is a typical autoimmune-mediated rheumatic disease presenting as a chronic synovitis in the joint. The chronic synovial inflammation is characterized by hyper-vascularity and extravasation of various immune-related cells to form lymphoid aggregates where an intimate cross-talk among innate and adaptive immune cells takes place. These interactions facilitate production of abundant proinflammatory cytokines, chemokines and growth factors for the proliferation/maturation/differentiation of B lymphocytes to become plasma cells. Finally, the autoantibodies against denatured immunoglobulin G (rheumatoid factors), EB virus nuclear antigens (EBNAs) and citrullinated protein (ACPAs) are produced to trigger the development of RA. Furthermore, it is documented that gene mutations, abnormal epigenetic regulation of peptidylarginine deiminase genes 2 and 4 (PADI2 and PADI4), and thereby the induced autoantibodies against PAD2 and PAD4 are implicated in ACPA production in RA patients. The aberrant expressions of non-coding RNAs (ncRNAs) including microRNAs (miRs) and long non-coding RNAs (lncRNAs) in the immune system undoubtedly derange the mRNA expressions of cytokines/chemokines/growth factors. In the present review, we will discuss in detail the expression of these ncRNAs and their target molecules participating in developing RA, and the potential biomarkers for the disease, its diagnosis, cardiovascular complications and therapeutic response. Finally, we propose some prospective investigations for unraveling the conundrums of rheumatoid pathogenesis.     

含钙高温锰系磷化膜性能的研究

向锰系磷化液中加入钙剂,制得含钙磷化膜。观察了磷化膜的微观形貌及生长过程,并测试了含钙磷化膜的成分、耐蚀性、膜重、生长速率、耐高温性及结合力。结果表明:含钙磷化膜表面由一些较大的、不规则的块状晶体和棒状晶体组成,含Mn、P、Fe及少量的Ca;含钙磷化膜的耐蚀性优于不含钙磷化膜的;含钙磷化膜能够耐受500℃的高温;含钙磷化膜与钢铁基体结合良好。     

Structural Basis for the Diminished Ligand Binding and Catalytic Ability of Human Fetal-Specific CYP3A7

Cytochrome P450 3A7 (CYP3A7) is a fetal/neonatal liver enzyme that participates in estriol synthesis, clearance of all-trans retinoic acid, and xenobiotic metabolism. Compared to the closely related major drug-metabolizing enzyme in adult liver, CYP3A4, the ligand binding and catalytic capacity of CYP3A7 are substantially reduced. To better understand the structural basis for these functional differences, the 2.15 Å crystal structure of CYP3A7 has been solved. Comparative analysis of CYP3A enzymes shows that decreased structural plasticity rather than the active site microenvironment defines the ligand binding ability of CYP3A7. In particular, a rotameric switch in the gatekeeping amino acid F304 triggers local and long-range rearrangements that transmit to the F-G fragment and alter its interactions with the I-E-D-helical core, resulting in a more rigid structure. Elongation of the β₃-β₄ strands, H-bond linkage in the substrate channel, and steric constraints in the C-terminal loop further increase the active site rigidity and limit conformational ensemble. Collectively, these structural distinctions lower protein plasticity and change the heme environment, which, in turn, could impede the spin-state transition essential for optimal reactivity and oxidation of substrates.     

聚合物对烷基苯磺酸钠与钙离子相互作用的影响

用浊度法研究了聚合物对烷基苯磺酸钠与钙离子的相互作用的影响,不同聚合物,如聚乙烯亚胺（ＰＥＩ）、聚乙烯吡咯烷酮（ＰＶＰ）和聚乙二醇（ＰＥＧ）在同一ｐＨ条件下的研究结果表明,ＰＥＩ比后两能更好地抑制ＬＡＳ钙盐的形成。同时一聚合物在不同ｐＨ条件下的研究发现,ＰＥＩ只有在一个适当的ｐＨ范围内,即自身具有适当的电荷密度,才能很好地发挥其上述作用。在体系中存在一定量的ＡＥＯ３条件下,也观察到了ＰＥＩ在抑制     

Calcium Signalling in Heart and Vessels: Role of Calmodulin and Downstream Calmodulin-Dependent Protein Kinases

Cardiovascular disease is the major cause of death worldwide. The success of medication and other preventive measures introduced in the last century have not yet halted the epidemic of cardiovascular disease. Although the molecular mechanisms of the pathophysiology of the heart and vessels have been extensively studied, the burden of ischemic cardiovascular conditions has risen to become a top cause of morbidity and mortality. Calcium has important functions in the cardiovascular system. Calcium is involved in the mechanism of excitation–contraction coupling that regulates numerous events, ranging from the production of action potentials to the contraction of cardiomyocytes and vascular smooth muscle cells. Both in the heart and vessels, the rise of intracellular calcium is sensed by calmodulin, a protein that regulates and activates downstream kinases involved in regulating calcium signalling. Among them is the calcium calmodulin kinase family, which is involved in the regulation of cardiac functions. In this review, we present the current literature regarding the role of calcium/calmodulin pathways in the heart and vessels with the aim to summarize our mechanistic understanding of this process and to open novel avenues for research.     

New Insights from Crystallographic Data: Diversity of Structural Motifs and Molecular Recognition Properties between Groups of IDO1 Structures

A large number of crystallographic structures of IDO1 in different ligand-bound and -unbound states have been disclosed over the last decade. Yet, only a few of them have been exploited for structure-based drug design (SBDD) campaigns. In this study, we analyzed the structural motifs and molecular-recognition properties of three groups of IDO1 structures: 1) structures containing the heme group and inhibitors in the catalytic site; 2) heme-free structures of IDO1; 3) substrate-bound structures of IDO1. The results suggest that unrelated conformations of the enzyme have been solved with different ligand-induced changes of secondary motifs that localize even in regions remote from the catalytic site. Moreover, the study identified an uncharted region of molecular-recognition space covered by IDO1 binding sites that could guide the selection of diverse structures for additional SBDD studies aimed at the identification of novel lead compounds with differentiated chemical scaffolds.