RESUMEN

Understanding the functions of metal ions in biological systems is crucial for many aspects of research, including deciphering their roles in diseases and potential therapeutic use. Structural information about the molecular or atomic details of these interactions, generated by methods like X-ray crystallography, cryo-electron microscopy, or nucleic magnetic resonance, frequently provides details that no other method can. As with any experimental method, they have inherent limitations that sometimes lead to an erroneous interpretation. This manuscript highlights different aspects of structural data available for metal-protein complexes. We examine the quality of modeling metal ion binding sites across different structure determination methods, where different kinds of errors stem from, and how they can impact correct interpretations and conclusions.

RESUMEN

Identifying and characterizing metal-binding sites (MBS) within macromolecular structures is imperative for elucidating their biological functions. CheckMyMetal (CMM) is a web based tool that facilitates the interactive validation of MBS in structures determined through X-ray crystallography and cryo-electron microscopy (cryo-EM). Recent updates to CMM have significantly enhanced its capability to efficiently handle large datasets generated from cryo-EM structural analyses. In this study, we address various challenges inherent in validating MBS within both X-ray and cryo-EM structures. Specifically, we examine the difficulties associated with accurately identifying metals and modeling their coordination environments by considering the ongoing reproducibility challenges in structural biology and the critical importance of well annotated, high-quality experimental data. CMM employs a sophisticated framework of rules rooted in the valence bond theory for MBS validation. We explore how CMM validation parameters correlate with the resolution of experimentally derived structures of macromolecules and their complexes. Additionally, we showcase the practical utility of CMM by analyzing a representative cryo-EM structure. Through a comprehensive examination of experimental data, we demonstrate the capability of CMM to advance MBS characterization and identify potential instances of metal misassignment.

Asunto(s)

Microscopía por Crioelectrón , Metales , Microscopía por Crioelectrón/métodos , Sitios de Unión , Cristalografía por Rayos X/métodos , Metales/química , Metales/metabolismo , Reproducibilidad de los Resultados , Modelos Moleculares , Programas Informáticos , Sustancias Macromoleculares/química

RESUMEN

Zinc is required for virtually all biological processes. In plasma, Zn2+ is predominantly transported by human serum albumin (HSA), which possesses two Zn2+-binding sites of differing affinities (sites A and B). Fatty acids (FAs) are also transported by HSA, with seven structurally characterized FA-binding sites (named FA1-FA7) known. FA binding inhibits Zn2+-HSA interactions, in a manner that can impact upon hemostasis and cellular zinc uptake, but the degree to which binding at specific FA sites contributes to this inhibition is unclear. Wild-type HSA and H9A, H67A, H247A, and Y150F/R257A/S287A (FA2-KO) mutant albumins were expressed in Pichia pastoris. Isothermal titration calorimetry studies revealed that the Zn2+-binding capacity at the high-affinity Zn2+ site (site A) was reduced in H67A and H247A mutants, with site B less affected. The H9A mutation decreased Zn2+ binding at the lower-affinity site, establishing His9 as a site B ligand. Zn2+ binding to HSA and H9A was compromised by palmitate, consistent with FA binding affecting site A. 13C-NMR experiments confirmed that the FA2-KO mutations prohibited FA binding at site FA2. Zn2+ binding to the FA2-KO mutant was unaffected by myristate, suggesting binding at FA2 is solely responsible for inhibition. Molecular dynamics studies identified the steric obstruction exerted by bound FA in site FA2, which impedes the conformational change from open (FA-loaded) to closed (FA-free) states, required for Zn2+ to bind at site A. The successful targeting of the FA2 site will aid functional studies exploring the interplay between circulating FA levels and plasma Zn2+ speciation in health and disease.

Asunto(s)

Ácidos Grasos , Albúmina Sérica Humana , Zinc , Zinc/metabolismo , Humanos , Sitios de Unión , Ácidos Grasos/metabolismo , Albúmina Sérica Humana/metabolismo , Albúmina Sérica Humana/química , Unión Proteica

RESUMEN

INTRODUCTION: Macromolecular X-ray crystallography and cryo-EM are currently the primary techniques used to determine the three-dimensional structures of proteins, nucleic acids, and viruses. Structural information has been critical to drug discovery and structural bioinformatics. The integration of artificial intelligence (AI) into X-ray crystallography has shown great promise in automating and accelerating the analysis of complex structural data, further improving the efficiency and accuracy of structure determination. AREAS COVERED: This review explores the relationship between X-ray crystallography and other modern structural determination methods. It examines the integration of data acquired from diverse biochemical and biophysical techniques with those derived from structural biology. Additionally, the paper offers insights into the influence of AI on X-ray crystallography, emphasizing how integrating AI with experimental approaches can revolutionize our comprehension of biological processes and interactions. EXPERT OPINION: Investing in science is crucially emphasized due to its significant role in drug discovery and advancements in healthcare. X-ray crystallography remains an essential source of structural biology data for drug discovery. Recent advances in biochemical, spectroscopic, and bioinformatic methods, along with the integration of AI techniques, hold the potential to revolutionize drug discovery when effectively combined with robust data management practices.

Asunto(s)

Inteligencia Artificial , Descubrimiento de Drogas , Humanos , Cristalografía por Rayos X , Descubrimiento de Drogas/métodos , Proteínas/química , Biología Computacional

RESUMEN

The overall quality of the experimentally determined structures contained in the PDB is exceptionally high, mainly due to the continuous improvement of model building and structural validation programs. Improving reproducibility on a large scale requires expanding the concept of validation in structural biology and all other disciplines to include a broader framework that encompasses the entire project. A successful approach to science requires diligent attention to detail and a focus on the future. An earnest commitment to data availability and reuse is essential for scientific progress, be that by human minds or artificial intelligence.

RESUMEN

Over the course of the pandemic caused by SARS-CoV-2, structural biologists have worked hand in hand with groups developing vaccines and treatments. However, relying solely on in vitro and clinical studies may be insufficient to guide vaccination and treatment developments, and other healthcare policies during virus mutations or peaks in infections and fatalities. Therefore, it is crucial to track statistical data related to the number of infections, deaths, and vaccinations in specific regions and present it in an easy-to-understand way.

RESUMEN

Metal ions bound to macromolecules play an integral role in many cellular processes. They can directly participate in catalytic mechanisms or be essential for the structural integrity of proteins and nucleic acids. However, their unique nature in macromolecules can make them difficult to model and refine, and a substantial portion of metal ions in the PDB are misidentified or poorly refined. CheckMyMetal (CMM) is a validation tool that has gained widespread acceptance as an essential tool for researchers working on metal-macromolecule complexes. CMM can be used during structure determination or to validate metal binding sites in structural models within the PDB. The functionalities of CMM have recently been greatly enhanced and provide researchers with additional information that can guide modeling decisions. The new version of CMM shows metals in the context of electron density maps and allows for on-the-fly refinement of metal binding sites. The improvements should increase the reproducibility of biomedical research. The web server is available at https://cmm.minorlab.org.

Asunto(s)

Metales , Proteínas , Sitios de Unión , Reproducibilidad de los Resultados , Modelos Moleculares , Proteínas/química , Metales/metabolismo , Iones

RESUMEN

Antimicrobial peptides (AMPs) are important components of innate immunity. Here, we report the antimicrobial properties of a peptide derived from the Male fertility factor kl2 (MFF-kl2) protein of Drosophila melanogaster, which was identified as a functional analog of the mammalian antibacterial chemerin-p4 peptide. The antimicrobial activity of multifunctional chemerin is mainly associated with a domain localized in the middle of the chemerin sequence, Val66-Pro85 peptide (chemerin-p4). Using bioinformatic tools, we found homologs of the chemerin-p4 peptide in the proteome of D. melanogaster. One of them is MFF-p1, which is a part of the MFF kl2 protein, encoded by the gene male fertility factor kl2 (kl-2) located on the long arm of the Y chromosome. The second detected peptide (Z-p1) is a part of the Zizimin protein belonging to DOCK family, which is involved in cellular signaling processes. After testing the antimicrobial properties of both peptides, we found that only MFF-p1 possesses these properties. Here, we demonstrate its antimicrobial potential both in vitro and in vivo after infecting D. melanogaster with bacteria. MFF-p1 strongly inhibits the viable counts of E. coli and B. subtilis after 2 h of treatment and disrupts bacterial cells. The expression of kl-2 is regulated by exposure to bacteria and by the circadian clock.

RESUMEN

For over 20 years, the OPLS-All Atom (OPLS-AA) force field has been efficiently used in molecular modelling studies of proteins, carbohydrates and nucleic acids. OPLS-AA is successfully applied in computer modelling of many organic compounds, including decane and shorter alkanes, but it fails when employed for longer linear alkanes, whose chemical structure corresponds to hydrocarbon tails in phospholipids constituting cellular membranes. There have been several attempts to address this problem. In this work, we compare the ability to reproduce various condensed phase properties by six distinct sets of force field parameters which can be assigned to phospholipid hydrocarbon chains. In this comparison, we include three alternative sets of the OPLS-AA force field, as well as the commonly used CHARMM C36, Slipids, and Berger lipids' parameters.

Asunto(s)

Alcanos/química , Modelos Químicos , Simulación de Dinámica Molecular , Difusión , Cinética , Membrana Dobles de Lípidos/química , Modelos Moleculares , Estructura Molecular , Fosfolípidos/química , Temperatura de Transición , Viscosidad , Volatilización

RESUMEN

Chemerin is a leukocyte attractant, adipokine, and antimicrobial protein abundantly produced in the skin epidermis. Despite the fact that most of the bactericidal activity present in human skin exudates is chemerin-dependent, just how chemerin shapes skin defenses remains obscure. Here we demonstrate that p4, a potent antimicrobial human chemerin peptide derivative, displays killing activity against pathogenic methicillin-resistant Staphylococcus aureus strains and suppresses microbial growth in a topical skin infection model. Mechanistically, we show that p4 homodimerization is required for maximal bactericidal activity and that an oxidative environment, such as at the skin surface, facilitates p4 disulfide bridge formation, required for the dimerization. p4 led to rapid damage of the bacterial internal membrane and inhibited the interaction between the membranous cytochrome bc1 complex and its redox partner, cytochrome c These results suggest that a chemerin p4-based defense strategy combats bacterial challenges at the skin surface.

Asunto(s)

Antibacterianos/farmacología , Quimiocinas/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Oligopéptidos/farmacología , Enfermedades Cutáneas Bacterianas/tratamiento farmacológico , Piel/efectos de los fármacos , Infecciones Estafilocócicas/tratamiento farmacológico , Animales , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Pruebas de Sensibilidad Microbiana , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Piel/metabolismo , Piel/microbiología , Enfermedades Cutáneas Bacterianas/metabolismo , Enfermedades Cutáneas Bacterianas/microbiología , Infecciones Estafilocócicas/metabolismo , Infecciones Estafilocócicas/microbiología

RESUMEN

In veterinary medicine, Staphylococcus aureus is associated with a range of mild to severe infections. The high density of livestock in intensive farming systems increases the risk of disease spread and hampers its control and measures of prevention, making S. aureus one of the most important animal pathogens. Multiple-locus variable-number tandem repeat fingerprinting (MLVF) has been successfully applied to the characterization of livestock-associated meticillin-resistant Staphylococcus aureus (MRSA) ST398 but not to the characterization of a wide range of other animal isolates. The objective of the current study was to examine the effectiveness of MLVF for studying S. aureus strains isolated from households, farms and exotic animals in three regions of Poland. MLVF, random amplification of polymorphic DNA (RAPD), spa typing and diagnostic microarrays were compared to determine the most suitable combination of methods for veterinary purposes. MLVF generated results consistent with host and geographic origins, reflecting population structures with a high concordance to spa typing results. MLVF has been proven to be a rapid, highly discriminatory and cost-effective method suitable for molecular typing in veterinary settings.

Asunto(s)

Animales Domésticos/microbiología , Técnicas de Tipificación Bacteriana , Dermatoglifia del ADN , Tipificación Molecular/veterinaria , Infecciones Estafilocócicas/veterinaria , Staphylococcus aureus/clasificación , Staphylococcus aureus/genética , Animales , Animales Exóticos , Antibacterianos/farmacología , Gatos/microbiología , Bovinos/microbiología , Pollos/microbiología , Análisis Costo-Beneficio , Dermatoglifia del ADN/economía , ADN Bacteriano/análisis , ADN Bacteriano/genética , Perros/microbiología , Equidae/microbiología , Composición Familiar , Genotipo , Ganado/microbiología , Análisis por Micromatrices , Pruebas de Sensibilidad Microbiana , Repeticiones de Minisatélite , Pan troglodytes/microbiología , Polonia/epidemiología , Conejos/microbiología , Infecciones Estafilocócicas/epidemiología , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/aislamiento & purificación

RESUMEN

Secretory leukocyte protease inhibitor (SLPI), a ∼12kDa nonglycosylated cationic protein, is emerging as an important regulator of innate and adaptive immunity and as a component of tissue regenerative programs. First described as an inhibitor of serine proteases such as neutrophil elastase, this protein is increasingly recognized as a molecule that benefits the host via its anti-proteolytic, anti-microbial and immunomodulatory activities. Here, we discuss the diverse functions of SLPI. Moreover, we review several novel layers of SLPI-mediated control that protect the host from excessive/dysregulated inflammation typical of infectious, allergic and autoinflammatory diseases and that support healing responses through affecting cell proliferation, differentiation and apoptosis.

Asunto(s)

Inhibidor Secretorio de Peptidasas Leucocitarias/inmunología , Inhibidor Secretorio de Peptidasas Leucocitarias/metabolismo , Animales , Autoinmunidad/inmunología , Humanos , Elastasa de Leucocito/inmunología , Elastasa de Leucocito/metabolismo , Cicatrización de Heridas/fisiología

RESUMEN

This review summarises high resolution studies on the interface of lamellar lipid bilayers composed of the most typical lipid molecules which constitute the lipid matrix of biomembranes. The presented results were obtained predominantly by computer modelling methods. Whenever possible, the results were compared with experimental results obtained for similar systems. The first and main section of the review is concerned with the bilayer-water interface and is divided into four subsections. The first describes the simplest case, where the interface consists only of lipid head groups and water molecules and focuses on interactions between the lipid heads and water molecules; the second describes the interface containing also mono- and divalent ions and concentrates on lipid-ion interactions; the third describes direct inter-lipid interactions. These three subsections are followed by a discussion on the network of direct and indirect inter-lipid interactions at the bilayer interface. The second section summarises recent computer simulation studies on the interactions of antibacterial membrane active compounds with various models of the bacterial outer membrane. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg.

Asunto(s)

Simulación por Computador , Membrana Dobles de Lípidos/química , Agua/química , Enlace de Hidrógeno , Fosfatidilcolinas/química , Fosfatidiletanolaminas/química , Fosfatidilserinas/química , Esfingomielinas/química

RESUMEN

Lipid A is the most chemically invariant part of lipopolysaccharides (LPS). Both lipid A and LPS constitute the external layer of the outer membrane of Gram-negative bacteria. E. coli-specific hexacyl lipid A (ECLA) forms stable bilayers in the presence of sodium or magnesium cations. To characterize biologically relevant properties of the ECLA bilayer, and in particular its water/membrane interface, 800 ns molecular dynamics (MD) simulations of fully hydrated bilayers made of ECLA at 50 °C (i.e., 6 °C above the main phase transition) were performed. The validation of the computer model for the ECLA bilayer was performed using available experimental data. The overall good agreement with the data was found. An ECLA molecule makes on average ∼1.3 ion-mediated bridges with neighboring lipid molecules. The average number of interlipid hydrogen bonds is 2.7. The abundance of such intermolecular links results in tight packing of ECLA molecules in the bilayer and explains the relatively small value of the surface area per lipid (1.515 nm(2)).

Asunto(s)

Proteínas de Escherichia coli/química , Lípido A/química , Membrana Dobles de Lípidos/química , Agua/química , Electrones , Escherichia coli , Enlace de Hidrógeno , Iones/química , Simulación de Dinámica Molecular , Conformación Proteica , Sodio/química , Temperatura , Factores de Tiempo

RESUMEN

The DMG-α library grants researchers in the field of computational biology, chemistry, and biophysics access to an open-sourced, easy to use, and intuitive software for performing fine-grained geometric analysis of molecular systems. The library is capable of computing power diagrams (weighted Voronoi diagrams) in three dimensions with 3D periodic boundary conditions, computing approximate projective 2D Voronoi diagrams on arbitrarily defined surfaces, performing shape properties recognition using α-shape theory and can do exact Solvent Accessible Surface Area (SASA) computation. The software is written mainly as a template-based C++ library for greater performance, but a rich Python interface (pydmga) is provided as a convenient way to manipulate the DMG-α routines. To illustrate possible applications of the DMG-α library, we present results of sample analyses which allowed to determine nontrivial geometric properties of two Escherichia coli-specific lipids as emerging from molecular dynamics simulations of relevant model bilayers.

Asunto(s)

Descubrimiento de Drogas/métodos , Programas Informáticos , Escherichia coli , Membrana Dobles de Lípidos/química , Simulación de Dinámica Molecular , Solventes/química , Interfaz Usuario-Computador

RESUMEN

Chemerin is a widely distributed multifunctional secreted protein implicated in immune cell migration, adipogenesis, osteoblastogenesis, angiogenesis, myogenesis, and glucose homeostasis. Chemerin message is regulated by nuclear receptor agonists, metabolic signaling proteins and intermediates, and proinflammatory cytokines. Following translation chemerin is secreted as an inactive pro-protein, and its secretion can be regulated depending on cell type. Chemerin bioactivity is largely dependent on carboxyl-terminal proteolytic processing and removal of inhibitory residues. Chemerin is abundant in human epidermis where it is well-placed to provide barrier protection. In host defense, chemerin plays dual roles as a broad spectrum antimicrobial protein and as a leukocyte attractant for macrophages, dendritic cells, and NK cells. Here we review the mechanisms underlying chemerin regulation and its function in host defense.

RESUMEN

OPLS All-Atom (OPLS/AA) is a generic all-atom force field which was fine-tuned to accurately reproduce condensed phase properties of organic liquids. Its application in modeling of lipid membranes is, however, limited mainly due to the inability to correctly describe phase behavior and organization of the hydrophobic core of the model lipid bilayers. Here we report new OPLS/AA parameters for n-pentadecane, methyl acetate, and dimethyl phosphate anion. For the new force field parameters, we show very good agreement between calculated and numerous reference data, including liquid density, enthalpy of vaporization, free energy of hydration, and selected transport properties. The new OPLS/AA parameters have been used in successful submicrosecond MD simulations of bilayers made of bacterial glycolipids whose results will be published elsewhere shortly.

Asunto(s)

Acetatos/química , Alcanos/química , Simulación de Dinámica Molecular , Compuestos Organofosforados/química , Conformación Molecular

RESUMEN

Chemerin, a chemoattractant ligand for chemokine-like receptor 1 (CMKLR1) is predicted to share similar tertiary structure with antibacterial cathelicidins. Recombinant chemerin has antimicrobial activity. Here we show that endogenous chemerin is abundant in human epidermis, and that inhibition of bacteria growth by exudates from organ cultures of primary human skin keratinocytes is largely chemerin-dependent. Using a panel of overlapping chemerin-derived synthetic peptides, we demonstrate that the antibacterial activity of chemerin is primarily mediated by Val(66)-Pro(85), which causes direct bacterial lysis. Therefore, chemerin is an antimicrobial agent in human skin.

Asunto(s)

Péptidos Catiónicos Antimicrobianos/inmunología , Quimiocinas/inmunología , Epidermis/inmunología , Epidermis/microbiología , Secuencia de Aminoácidos , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/genética , Células Cultivadas , Quimiocinas/química , Quimiocinas/genética , Escherichia coli/inmunología , Escherichia coli/patogenicidad , Humanos , Péptidos y Proteínas de Señalización Intercelular , Queratinocitos/inmunología , Queratinocitos/microbiología , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología

RESUMEN

Lutein is present in the human retina and lens, where it plays a protective role. As lutein is associated with the lipid matrix of biomembranes, the role depends on its membrane location. Experimental studies predicted two orientations of lutein in a phosphatidylcholine (PC) bilayer: vertical and horizontal. Using a molecular dynamics simulation, we observed, in two different PC bilayers, both orientations of lutein, and in each bilayer, a single change from vertical to horizontal orientation or vice versa. Both orientations were stabilized by hydrogen bonding of lutein OH groups with mainly carbonyl but also phosphate oxygen atoms of PC.

Asunto(s)

Membrana Dobles de Lípidos/química , Luteína/química , Enlace de Hidrógeno , Modelos Moleculares , Simulación de Dinámica Molecular , Fosfatidilcolinas/química

RESUMEN

Dethiobiotin synthetase (DTBS) is involved in the biosynthesis of biotin in bacteria, fungi, and plants. As humans lack this pathway, DTBS is a promising antimicrobial drug target. We determined structures of DTBS from Helicobacter pylori (hpDTBS) bound with cofactors and a substrate analog, and described its unique characteristics relative to other DTBS proteins. Comparison with bacterial DTBS orthologs revealed considerable structural differences in nucleotide recognition. The C-terminal region of DTBS proteins, which contains two nucleotide-recognition motifs, differs greatly among DTBS proteins from different species. The structure of hpDTBS revealed that this protein is unique and does not contain a C-terminal region containing one of the motifs. The single nucleotide-binding motif in hpDTBS is similar to its counterpart in GTPases; however, isothermal titration calorimetry binding studies showed that hpDTBS has a strong preference for ATP. The structural determinants of ATP specificity were assessed with X-ray crystallographic studies of hpDTBS·ATP and hpDTBS·GTP complexes. The unique mode of nucleotide recognition in hpDTBS makes this protein a good target for H. pylori-specific inhibitors of the biotin synthesis pathway.

Asunto(s)

Proteínas Bacterianas/química , Ligasas de Carbono-Nitrógeno/química , Helicobacter pylori/enzimología , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Sitios de Unión , Ligasas de Carbono-Nitrógeno/metabolismo , Cristalografía por Rayos X , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Alineación de Secuencia , Especificidad por Sustrato