X-Ray Crystallography for Macromolecular Complexes.

X-ray crystallography has for most of the last century been the standard technique to determine the high-resolution structure of biological macromolecules, including multi-subunit protein-protein and protein-nucleic acids as large as the ribosome and viruses. As such, the successful application of X-ray crystallography to many biological problems revolutionized biology and biomedicine by solving the structures of small molecules and vitamins, peptides and proteins, DNA and RNA molecules, and many complexes-affording a detailed knowledge of the structures that clarified biological and chemical mechanisms, conformational changes, interactions, catalysis and the biological processes underlying DNA replication, translation, and protein synthesis. Now reaching well into the first quarter of the twenty-first century, X-ray crystallography shares the structural biology stage with cryo-electron microscopy and other innovative structure determination methods, as relevant and central to our understanding of biological function and structure as ever. In this chapter, we provide an overview of modern X-ray crystallography and how it interfaces with other mainstream structural biology techniques, with an emphasis on macromolecular complexes.

Protein-Protein Binding Kinetics by Biolayer Interferometry.

The specific kinetics and thermodynamics of protein-protein interactions underlie the molecular mechanisms of cellular functions; hence the characterization of these interaction parameters is central to the quantitative understanding of physiological and pathological processes. Many methods have been developed to study protein-protein interactions, which differ in various features including the interaction detection principle, the sensitivity, whether the method operates in vivo, in vitro, or in silico, the temperature control, the use of labels, immobilization, the amount of sample required, the number of measurements that can be accomplished simultaneously, or the cost. Bio-Layer Interferometry (BLI) is a label-free biophysical method to measure the kinetics of protein-protein interactions. Label-free interaction assays are a broad family of methods that do not require protein modifications (other than immobilization) or labels such as fusions with fluorescent proteins or transactivating domains or chemical modifications like biotinylation or reaction with radionuclides. Besides BLI, other label-free techniques that are widely used for determining protein-protein interactions include surface plasmon resonance (SPR), thermophoresis, and isothermal titration calorimetry (ITC), among others.

Decoding anaphylatoxins: unveiling the molecular mechanisms of complement receptor activation and signaling.

Recent advances in cryo-electron microscopy (Cryo-EM) have revolutionized our understanding of the complement C5a/C3a receptors that are crucial in inflammation. A recent report by Yadav et al. has elucidated the activation, ligand binding, selectivity, and signaling bias of these receptors, thereby enhancing structure-guided drug discovery. This paves the way for more effective anti-inflammatory therapies that target these receptors with unprecedented precision.

Structural biology of complement receptors.

The complement system plays crucial roles in a wide breadth of immune and inflammatory processes and is frequently cited as an etiological or aggravating factor in many human diseases, from asthma to cancer. Complement receptors encompass at least eight proteins from four structural classes, orchestrating complement-mediated humoral and cellular effector responses and coordinating the complex cross-talk between innate and adaptive immunity. The progressive increase in understanding of the structural features of the main complement factors, activated proteolytic fragments, and their assemblies have spurred a renewed interest in deciphering their receptor complexes. In this review, we describe what is currently known about the structural biology of the complement receptors and their complexes with natural agonists and pharmacological antagonists. We highlight the fundamental concepts and the gray areas where issues and problems have been identified, including current research gaps. We seek to offer guidance into the structural biology of the complement system as structural information underlies fundamental and therapeutic research endeavors. Finally, we also indicate what we believe are potential developments in the field.

The structure of Leptospira interrogans GAPDH sheds light into an immunoevasion factor that can target the anaphylatoxin C5a of innate immunity.

Leptospirosis is a neglected worldwide zoonosis involving farm animals and domestic pets caused by the Gram-negative spirochete Leptospira interrogans. This bacterium deploys a variety of immune evasive mechanisms, some of them targeted at the complement system of the host's innate immunity. In this work, we have solved the X-ray crystallographic structure of L. interrogans glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to 2.37-Å resolution, a glycolytic enzyme that has been shown to exhibit moonlighting functions that potentiate infectivity and immune evasion in various pathogenic organisms. Besides, we have characterized the enzyme's kinetic parameters toward the cognate substrates and have proven that the two natural products anacardic acid and curcumin are able to inhibit L. interrogans GAPDH at micromolar concentration through a noncompetitive inhibition modality. Furthermore, we have established that L. interrogans GAPDH can interact with the anaphylatoxin C5a of human innate immunity in vitro using bio-layer interferometry and a short-range cross-linking reagent that tethers free thiol groups in protein complexes. To shed light into the interaction between L. interrogans GAPDH and C5a, we have also carried out cross-link guided protein-protein docking. These results suggest that L. interrogans could be placed in the growing list of bacterial pathogens that exploit glycolytic enzymes as extracellular immune evasive factors. Analysis of the docking results indicates a low affinity interaction that is consistent with previous evidence, including known binding modes of other α-helical proteins with GAPDH. These findings allow us to propose L. interrogans GAPDH as a potential immune evasive factor targeting the complement system.

Soluble endoglin reduces thrombus formation and platelet aggregation via interaction with αIIbß3 integrin.

BACKGROUND: The circulating form of human endoglin (sEng) is a cleavage product of membrane-bound endoglin present on endothelial cells. Because sEng encompasses an RGD motif involved in integrin binding, we hypothesized that sEng would be able to bind integrin αIIbß3, thereby compromising platelet binding to fibrinogen and thrombus stability. METHODS: In vitro human platelet aggregation, thrombus retraction, and secretion-competition assays were performed in the presence of sEng. Surface plasmon resonance (SPR) binding and computational (docking) analyses were carried out to evaluate protein-protein interactions. A transgenic mouse overexpressing human sEng (hsEng+) was used to measure bleeding/rebleeding, prothrombin time (PT), blood stream, and embolus formation after FeCl3-induced injury of the carotid artery. RESULTS: Under flow conditions, supplementation of human whole blood with sEng led to a smaller thrombus size. sEng inhibited platelet aggregation and thrombus retraction, interfering with fibrinogen binding, but did not affect platelet activation. SPR binding studies demonstrated that the specific interaction between αIIbß3 and sEng and molecular modeling showed a good fitting between αIIbß3 and sEng structures involving the endoglin RGD motif, suggesting the possible formation of a highly stable αIIbß3/sEng. hsEng+ mice showed increased bleeding time and number of rebleedings compared to wild-type mice. No differences in PT were denoted between genotypes. After FeCl3 injury, the number of released emboli in hsEng+ mice was higher and the occlusion was slower compared to controls. CONCLUSIONS: Our results demonstrate that sEng interferes with thrombus formation and stabilization, likely via its binding to platelet αIIbß3, suggesting its involvement in primary hemostasis control.

The Hidden Side of Complement Regulator C4BP: Dissection and Evaluation of Its Immunomodulatory Activity.

C4b-binding protein (C4BP) is a well-known regulator of the complement system that holds additional and important activities unrelated to complement inhibition. Recently, we have described a novel immunomodulatory activity in the minor C4BP(ß-) isoform directly acting over inflammatory phagocytes. Here we show that incorporation of the ß-chain to the C4BP α-chain oligomer interferes with this immunomodulatory activity of C4BP. Moreover, an oligomeric form including only the complement control protein 6 (CCP6) domain of the C4BP α-chain (PRP6-HO7) is sufficient to "reprogram" monocyte-derived DCs (Mo-DCs) from a pro-inflammatory and immunogenic phenotype to an anti-inflammatory and tolerogenic state. PRP6-HO7 lacks complement regulatory activity but retains full immunomodulatory activity over inflammatory Mo-DCs induced by TLRs, characterized by downregulation of relevant surface markers such as CD83, HLA-DR, co-stimulatory molecules such as CD86, CD80 and CD40, and pro-inflammatory cytokines such as IL-12 and TNF-α. Furthermore, PRP6-HO7-treated Mo-DCs shows increased endocytosis, significantly reduced CCR7 expression and CCL21-mediated chemotaxis, and prevents T cell alloproliferation. Finally, PRP6-HO7 shows also full immunomodulatory activity over Mo-DCs isolated from lupus nephritis patients with active disease, even without further pro-inflammatory stimulation. Therefore PRP6-HO7, retaining the immunomodulatory activity of C4BP(ß-) and lacking its complement regulatory activity, might represent a promising and novel alternative to treat autoimmune diseases.

The crystal structure of iC3b-CR3 αI reveals a modular recognition of the main opsonin iC3b by the CR3 integrin receptor.

Complement activation on cell surfaces leads to the massive deposition of C3b, iC3b, and C3dg, the main complement opsonins. Recognition of iC3b by complement receptor type 3 (CR3) fosters pathogen opsonophagocytosis by macrophages and the stimulation of adaptive immunity by complement-opsonized antigens. Here, we present the crystallographic structure of the complex between human iC3b and the von Willebrand A inserted domain of the α chain of CR3 (αI). The crystal contains two composite interfaces for CR3 αI, encompassing distinct sets of contiguous macroglobulin (MG) domains on the C3c moiety, MG1-MG2 and MG6-MG7 domains. These composite binding sites define two iC3b-CR3 αI complexes characterized by specific rearrangements of the two semi-independent modules, C3c moiety and TED domain. Furthermore, we show the structure of iC3b in a physiologically-relevant extended conformation. Based on previously available data and novel insights reported herein, we propose an integrative model that reconciles conflicting facts about iC3b structure and function and explains the molecular basis for iC3b selective recognition by CR3 on opsonized surfaces.

Generation of a Soluble Form of Human Endoglin Fused to Green Fluorescent Protein.

Endoglin (Eng, CD105) is a type I membrane glycoprotein that functions in endothelial cells as an auxiliary receptor for transforming growth factor ß (TGF-ß)/bone morphogenetic protein (BMP) family members and as an integrin ligand, modulating the vascular pathophysiology. Besides the membrane-bound endoglin, there is a soluble form of endoglin (sEng) that can be generated by the action of the matrix metalloproteinase (MMP)-14 or -12 on the juxtamembrane region of its ectodomain. High levels of sEng have been reported in patients with preeclampsia, hypercholesterolemia, atherosclerosis and cancer. In addition, sEng is a marker of cardiovascular damage in patients with hypertension and diabetes, plays a pathogenic role in preeclampsia, and inhibits angiogenesis and tumor proliferation, migration, and invasion in cancer. However, the mechanisms of action of sEng have not yet been elucidated, and new tools and experimental approaches are necessary to advance in this field. To this end, we aimed to obtain a fluorescent form of sEng as a new tool for biological imaging. Thus, we cloned the extracellular domain of endoglin in the pEGFP-N1 plasmid to generate a fusion protein with green fluorescent protein (GFP), giving rise to pEGFP-N1/Eng.EC. The recombinant fusion protein was characterized by transient and stable transfections in CHO-K1 cells using fluorescence microscopy, SDS-PAGE, immunodetection, and ELISA techniques. Upon transfection with pEGFP-N1/Eng.EC, fluorescence was readily detected in cells, indicating that the GFP contained in the recombinant protein was properly folded into the cytosol. Furthermore, as evidenced by Western blot analysis, the secreted fusion protein yielded the expected molecular mass and displayed a specific fluorescent signal. The fusion protein was also able to bind to BMP9 and BMP10 in vitro. Therefore, the construct described here could be used as a tool for functional in vitro studies of the extracellular domain of endoglin.

Using a partial atomic model from medium-resolution cryo-EM to solve a large crystal structure.

Medium-resolution cryo-electron microscopy maps, in particular when they include a significant number of α-helices, may allow the building of partial models that are useful for molecular-replacement searches in large crystallographic structures when the structures of homologs are not available and experimental phasing has failed. Here, as an example, the solution of the structure of a bacteriophage portal using a partial 30% model built into a 7.8 Šresolution cryo-EM map is shown. Inspection of the self-rotation function allowed the correct oligomerization state to be determined, and density-modification procedures using rotation matrices and a mask based on the cryo-EM structure were critical for solving the structure. A workflow is described that may be applicable to similar cases and this strategy is compared with direct use of the cryo-EM map for molecular replacement.

Structures of T7 bacteriophage portal and tail suggest a viral DNA retention and ejection mechanism.

Double-stranded DNA bacteriophages package their genome at high pressure inside a procapsid through the portal, an oligomeric ring protein located at a unique capsid vertex. Once the DNA has been packaged, the tail components assemble on the portal to render the mature infective virion. The tail tightly seals the ejection conduit until infection, when its interaction with the host membrane triggers the opening of the channel and the viral genome is delivered to the host cell. Using high-resolution cryo-electron microscopy and X-ray crystallography, here we describe various structures of the T7 bacteriophage portal and fiber-less tail complex, which suggest a possible mechanism for DNA retention and ejection: a portal closed conformation temporarily retains the genome before the tail is assembled, whereas an open portal is found in the tail. Moreover, a fold including a seven-bladed ß-propeller domain is described for the nozzle tail protein.

High-Throughput Protein Production in Yeast.

Yeasts are versatile single-celled fungi that grow to high cell densities on inexpensive media. With well-studied genetics and metabolism and a wealth of knowledge available about their propagation and growth in academic as well as industrial settings, yeasts have long been used for recombinant protein production of isolated proteins and multisubunit complexes. They can be easily adapted to high-throughput protein expression pipelines. Importantly, the outcome from small-scale expression evaluations in high-throughput mode is scalable to laboratory and industrial scales using well-established procedures. In this chapter, we offer a state-of-the-art perspective on currently available high-throughput pipelines for protein production in S. cerevisiae and P. pastoris and discuss future challenges and avenues for improvement.

High-Throughput Micro-Characterization of RNA-Protein Interactions.

Many cellular processes depend on and are regulated by nucleic acid-protein interactions. In particular, RNA-binding proteins (RBPs) are involved in transcription, translation, modulating RNA polymerase activity, and stabilizing protein-RNA complexes. Furthermore, RBPs participate in the development of pathologies such as cancer and viral infections, and their dysfunction leads to mutations and the aberrant expression of noncoding RNAs. Therefore, the study of RNA-protein interactions represents a central issue for biology and biomedicine. While many valuable insights have been obtained from electrophoretic mobility shift assays (EMSA) and immunoprecipitation (IP), these standard methods suffer from two main limitations: insufficient sensitivity to capture low concentration RBP-RNA complexes in vitro and identification of interactions in vivo. In recent years, high-throughput (HTP) platforms have emerged that combine methodological improvements over conventional techniques with more sensitive detection systems, thereby catalyzing the simultaneous probing and analysis of a vast amount of RBP-RNA interactions by cellular proteomics and interactomics approaches. In this chapter, we summarize a selection of state-of-the-art in vitro, in vivo, and computational HTP platforms for the discovery and characterization of RNA-protein interactions. We also reflect on the wealth of information obtained by the structural analysis of RBPs and their RNA-binding domains as a valuable resource for the rational design and implementation of new RNA-binding discovery platforms.

Peroxisomal catalases from the yeasts Pichia pastoris and Kluyveromyces lactis as models for oxidative damage in higher eukaryotes.

Catalases are among the main scavengers of reactive oxygen species (ROS) present in the peroxisome, thereby preventing oxidative cellular and tissular damage. In human, multiple diseases are associated with malfunction of these organelles, which causes accumulation of ROS species and consequently the inefficient detoxification of cells. Despite intense research, much remains to be clarified about the precise molecular role of catalase in cellular homeostasis. Yeast peroxisomes and their peroxisomal catalases have been used as eukaryotic models for oxidative metabolism, ROS generation and detoxification, and associated pathologies. In order to provide reliable models for oxidative metabolism research, we have determined the high-resolution crystal structures of peroxisomal catalase from two important biotechnology and basic biology yeast models, Pichia pastoris and Kluyveromyces lactis. We have performed an extensive functional, biochemical and stability characterization of both enzymes in order to establish their differential activity profiles. Furthermore, we have analyzed the role of the peroxisomal catalase under study in the survival of yeast to oxidative burst challenges combining methanol, water peroxide, and sodium chloride. Interestingly, whereas catalase activity was induced 200-fold upon challenging the methylotrophic P. pastoris cells with methanol, the increase in catalase activity in the non-methylotrophic K. lactis was only moderate. The inhibitory effect of sodium azide and ß-mercaptoethanol over both catalases was analyzed, establishing IC50 values for both compounds that are consistent with an elevated resistance of both enzymes toward these inhibitors. Structural comparison of these two novel catalase structures allows us to rationalize the differential susceptibility to inhibitors and oxidative bursts. The inherent worth and validity of the P. pastoris and K. lactis yeast models for oxidative damage will be strengthened by the availability of reliable structural-functional information on these enzymes, which are central to our understanding of peroxisomal response toward oxidative stress.

The Antimicrobials Anacardic Acid and Curcumin Are Not-Competitive Inhibitors of Gram-Positive Bacterial Pathogenic Glyceraldehyde-3-Phosphate Dehydrogenase by a Mechanism Unrelated to Human C5a Anaphylatoxin Binding.

The ubiquitous and highly abundant glycolytic enzyme D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is pivotal for the energy and carbon metabolism of most organisms, including human pathogenic bacteria. For bacteria that depend mostly on glycolysis for survival, GAPDH is an attractive target for inhibitor discovery. The availability of high-resolution structures of GAPDH from various pathogenic bacteria is central to the discovery of new antibacterial compounds. We have determined the X-ray crystal structures of two new GAPDH enzymes from Gram-positive bacterial pathogens, Streptococcus pyogenes and Clostridium perfringens. These two structures, and the recent structure of Atopobium vaginae GAPDH, reveal details in the active site that can be exploited for the design of novel inhibitors based on naturally occurring molecules. Two such molecules, anacardic acid and curcumin, have been found to counter bacterial infection in clinical settings, although the cellular targets responsible for their antimicrobial properties remain unknown. We show that both anacardic acid and curcumin inhibit GAPDH from two bacterial pathogens through uncompetitive and non-competitive mechanisms, suggesting GAPDH as a relevant pharmaceutical target for antibacterial development. Inhibition of GAPDH by anacardic acid and curcumin seems to be unrelated to the immune evasion function of pathogenic bacterial GAPDH, since neither natural compound interfere with binding to the human C5a anaphylatoxin.

Pathogens' toolbox to manipulate human complement.

The surveillance and pathogen fighting functions of the complement system have evolved to protect mammals from life-threatening infections. In turn, pathogens have developed complex molecular mechanisms to subvert, divert and evade the effector functions of the complement. The study of complement immunoevasion by pathogens sheds light on their infection drivers, knowledge that is essential to implement therapies. At the same time, complement evasion also acts as a discovery ground that reveals important aspects of how complement works under physiological conditions. In recent years, complex interrelationships between infection insults and the onset of autoimmune and complement dysregulation diseases have led to propose that encounters with pathogens can act as triggering factors for disease. The correct management of these diseases involves the recognition of their triggering factors and the development and administration of complement-associated molecular therapies. Even more recently, unsuspected proteins from pathogens have been shown to possess moonlighting functions as virulence factors, raising the possibility that behind the first line of virulence factors there be many more pathogen proteins playing secondary, helping and supporting roles for the pathogen to successfully establish infections. In an era where antibiotics have a progressively reduced effect on the management and control of infectious diseases worldwide, knowledge on the mechanisms of pathogenic invasion and evasion look more necessary and pressing than ever.

Protein topology determines substrate-binding mechanism in homologous enzymes.

During evolution, some homologs proteins appear with different connectivity between secondary structures (different topology) but conserving the tridimensional arrangement of them (same architecture). These events can produce two types of arrangements; circular permutation or non-cyclic permutations. The first one results in the N and C terminus transferring to a different position on a protein sequence while the second refers to a more complex arrangement of the structural elements. In ribokinase superfamily, two different topologies can be identified, which are related to each other as a non-cyclic permutation occurred during the evolution. Interestingly, this change in topology is correlated with the nucleotide specificity of its members. Thereby, the connectivity of the secondary elements allows us to distinguish an ATP-dependent and an ADP-dependent topology. Here we address the impact of introducing the topology of a homologous ATP-dependent kinase in an ADP-dependent kinase (Thermococcus litoralis glucokinase) in the structure, nucleotide specificity, and substrate binding order of the engineered enzyme. Structural evidence demonstrates that rewiring the topology of TlGK leads to an active and soluble enzyme without modifications on its three-dimensional architecture. The permuted enzyme (PerGK) retains the nucleotide preference of the parent TlGK enzyme but shows a change in the substrate binding order. Our results illustrate how the rearrangement of the protein folding topology during the evolution of the ribokinase superfamily enzymes may have dictated the substrate-binding order in homologous enzymes of this superfamily.

Insights into the inhibited form of the redox-sensitive SufE-like sulfur acceptor CsdE.

Sulfur trafficking in living organisms relies on transpersulfuration reactions consisting in the enzyme-catalyzed transfer of S atoms via activated persulfidic S across protein-protein interfaces. The recent elucidation of the mechanistic basis for transpersulfuration in the CsdA-CsdE model system has paved the way for a better understanding of its role under oxidative stress. Herein we present the crystal structure of the oxidized, inactivated CsdE dimer at 2.4 Å resolution. The structure sheds light into the activation of the Cys61 nucleophile on its way from a solvent-secluded position in free CsdE to a fully extended conformation in the persulfurated CsdA-CsdE complex. Molecular dynamics simulations of available CsdE structures allow to delineate the sequence of conformational changes underwent by CsdE and to pinpoint the key role played by the deprotonation of the Cys61 thiol. The low-energy subunit orientation in the disulfide-bridged CsdE dimer demonstrates the likely physiologic relevance of this oxidative dead-end form of CsdE, suggesting that CsdE could act as a redox sensor in vivo.

Protein-tRNA Agarose Gel Retardation Assays for the Analysis of the N 6-threonylcarbamoyladenosine TcdA Function.

We demonstrate methods for the expression and purification of tRNA(UUU) in Escherichia coli and the analysis by gel retardation assays of the binding of tRNA(UUU) to TcdA, an N6-threonylcarbamoyladenosine (t6A) dehydratase, which cyclizes the threonylcarbamoyl side chain attached to A37 in the anticodon stem loop (ASL) of tRNAs to cyclic t6A (ct6A). Transcription of the synthetic gene encoding tRNA(UUU) is induced in E. coli with 1 mM isopropyl ß-D-1-thiogalactopyranoside (IPTG) and the cells containing tRNA are harvested 24 h post-induction. The RNA fraction is purified using the acid phenol extraction method. Pure tRNA is obtained by a gel filtration chromatography that efficiently separates the small-sized tRNA molecules from larger intact or fragmented nucleic acids. To analyze TcdA binding to tRNA(UUU), TcdA is mixed with tRNA(UUU) and separated on a native agarose gel at 4 °C. The free tRNA(UUU) migrates faster, while the TcdA-tRNA(UUU) complexes undergo a mobility retardation that can be observed upon staining of the gel. We demonstrate that TcdA is a tRNA(UUU)-binding enzyme. This gel retardation assay can be used to study TcdA mutants and the effects of additives and other proteins on binding.