The structure of the GM-CSF receptor complex reveals a distinct mode of cytokine receptor activation.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic cytokine that controls the production and function of blood cells, is deregulated in clinical conditions such as rheumatoid arthritis and leukemia, yet offers therapeutic value for other diseases. Its receptors are heterodimers consisting of a ligand-specific alpha subunit and a betac subunit that is shared with the interleukin (IL)-3 and IL-5 receptors. How signaling is initiated remains an enigma. We report here the crystal structure of the human GM-CSF/GM-CSF receptor ternary complex and its assembly into an unexpected dodecamer or higher-order complex. Importantly, mutagenesis of the GM-CSF receptor at the dodecamer interface and functional studies reveal that dodecamer formation is required for receptor activation and signaling. This unusual form of receptor assembly likely applies also to IL-3 and IL-5 receptors, providing a structural basis for understanding their mechanism of activation and for the development of therapeutics.

Structural basis of the proteolytic and chaperone activity of Chlamydia trachomatis CT441.

Chlamydia trachomatis is the most prevalent cause of preventable blindness worldwide and a major reason for infectious infertility in females. Several bacterial factors have been implicated in the pathogenesis of C. trachomatis. Combining structural and mutational analysis, we have shown that the proteolytic function of CT441 depends on a conserved Ser/Lys/Gln catalytic triad and a functional substrate-binding site within a flexible PDZ (postsynaptic density of 95 kDa, discs large, and zonula occludens) domain. Previously, it has been suggested that CT441 is involved in modulating estrogen signaling responses of the host cell. Our results show that although in vitro CT441 exhibits proteolytic activity against SRAP1, a coactivator of estrogen receptor α, CT441-mediated SRAP1 degradation is not observed during the intracellular developmental cycle before host cells are lysed and infectious chlamydiae are released. Most compellingly, we have newly identified a chaperone activity of CT441, indicating a role of CT441 in prokaryotic protein quality control processes.

Structural and mechanistic analysis of the membrane-embedded glycosyltransferase WaaA required for lipopolysaccharide synthesis.

WaaA is a key enzyme in the biosynthesis of LPS, a critical component of the outer envelope of Gram-negative bacteria. Embedded in the cytoplasmic face of the inner membrane, WaaA catalyzes the transfer of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) to the lipid A precursor of LPS. Here we present crystal structures of the free and CMP-bound forms of WaaA from Aquifex aeolicus, an ancient Gram-negative hyperthermophile. These structures reveal details of the CMP-binding site and implicate a unique sequence motif (GGS/TX(5)GXNXLE) in Kdo binding. In addition, a cluster of highly conserved amino acid residues was identified which represents the potential membrane-attachment and acceptor-substrate binding site of WaaA. A series of site-directed mutagenesis experiments revealed critical roles for glycine 30 and glutamate 31 in Kdo transfer. Our results provide the structural basis of a critical reaction in LPS biosynthesis and allowed the development of a detailed model of the catalytic mechanism of WaaA.

Architecture and regulation of HtrA-family proteins involved in protein quality control and stress response.

Protein quality control is vital for all living cells and sophisticated molecular mechanisms have evolved to prevent the excessive accumulation of unfolded proteins. High-temperature requirement A (HtrA) proteases have been identified as important ATP-independent quality-control factors in most species. HtrA proteins harbor a serine-protease domain and at least one peptide-binding PDZ domain to ensure efficient removal of misfolded or damaged proteins. One distinctive property of HtrAs is their ability to assemble into complex oligomers. Whereas all examined HtrAs are capable of forming pyramidal 3-mers, higher-order complexes consisting of up to 24 molecules have been reported. Tight control of chaperone and protease function is of pivotal importance in preventing deleterious HtrA-protease activity. In recent years, structural biology provided detailed insights into the molecular basis of the regulatory mechanisms, which include unique intramolecular allosteric signaling cascades and the dynamic switching of oligomeric states of HtrA proteins. Based on these results, functional models for many family members have been developed. The HtrA protein family represents a remarkable example of how structural and functional diversity is attained from the assembly of simple molecular building blocks.

The Legionella HtrA homologue DegQ is a self-compartmentizing protease that forms large 12-meric assemblies.

Proteases of the HtrA family are key factors dealing with folding stress in the periplasmatic compartment of prokaryotes. In Escherichia coli, the well-characterized HtrA family members DegS and DegP counteract the accumulation of unfolded outer-membrane proteins under stress conditions. Whereas DegS serves as a folding-stress sensor, DegP is a chaperone-protease facilitating refolding or degradation of defective outer-membrane proteins. Here, we report the 2.15-Å-resolution crystal structure of the second major chaperone-protease of the periplasm, DegQ from Legionella fallonii. DegQ assembles into large, cage-like 12-mers that form independently of unfolded substrate proteins. We provide evidence that 12-mer formation is essential for the degradation of substrate proteins but not for the chaperone activity of DegQ. In the current model for the regulation of periplasmatic chaperone-proteases, 6-meric assemblies represent important protease-resting states. However, DegQ is unable to form such 6-mers, suggesting divergent regulatory mechanisms for DegQ and DegP. To understand how the protease activity of DegQ is controlled, we probed its functional properties employing designed protein variants. Combining crystallographic, biochemical, and mutagenic data, we present a mechanistic model that suggests how protease activity of DegQ 12-mers is intrinsically regulated and how deleterious proteolysis by free DegQ 3-mers is prevented. Our study sheds light on a previously uncharacterized component of the prokaryotic stress-response system with implications for other members of the HtrA family.

Exoerythrocytic Plasmodium parasites secrete a cysteine protease inhibitor involved in sporozoite invasion and capable of blocking cell death of host hepatocytes.

Plasmodium parasites must control cysteine protease activity that is critical for hepatocyte invasion by sporozoites, liver stage development, host cell survival and merozoite liberation. Here we show that exoerythrocytic P. berghei parasites express a potent cysteine protease inhibitor (PbICP, P. berghei inhibitor of cysteine proteases). We provide evidence that it has an important function in sporozoite invasion and is capable of blocking hepatocyte cell death. Pre-incubation with specific anti-PbICP antiserum significantly decreased the ability of sporozoites to infect hepatocytes and expression of PbICP in mammalian cells protects them against peroxide- and camptothecin-induced cell death. PbICP is secreted by sporozoites prior to and after hepatocyte invasion, localizes to the parasitophorous vacuole as well as to the parasite cytoplasm in the schizont stage and is released into the host cell cytoplasm at the end of the liver stage. Like its homolog falstatin/PfICP in P. falciparum, PbICP consists of a classical N-terminal signal peptide, a long N-terminal extension region and a chagasin-like C-terminal domain. In exoerythrocytic parasites, PbICP is posttranslationally processed, leading to liberation of the C-terminal chagasin-like domain. Biochemical analysis has revealed that both full-length PbICP and the truncated C-terminal domain are very potent inhibitors of cathepsin L-like host and parasite cysteine proteases. The results presented in this study suggest that the inhibitor plays an important role in sporozoite invasion of host cells and in parasite survival during liver stage development by inhibiting host cell proteases involved in programmed cell death.

Longitudinal Tracking of Immune Responses in COVID-19 Convalescents Reveals Absence of Neutralization Activity Against Omicron and Staggered Impairment to Other SARS-CoV-2 Variants of Concern.

Evaluating long-term protection against SARS-CoV-2 variants of concern in convalescing individuals is of high clinical relevance. In this prospective study of a cohort of 46 SARS-CoV-2 patients infected with the Wuhan strain of SARS-CoV-2 we longitudinally analyzed changes in humoral and cellular immunity upon early and late convalescence. Antibody neutralization capacity was measured by surrogate virus neutralization test and cellular responses were investigated with 31-colour spectral flow cytometry. Spike-specific, isotype-switched B cells developed already during the disease phase, showed a memory phenotype and did not decrease in numbers even during late convalescence. Otherwise, no long-lasting perturbations of the immune compartment following COVID-19 clearance were observed. During convalescence anti-Spike (S1) IgG antibodies strongly decreased in all patients. We detected neutralizing antibodies against the Wuhan strain as well as the Alpha and Delta but not against the Beta, Gamma or Omicron variants for up to 7 months post COVID-19. Furthermore, correlation analysis revealed a strong association between sera anti-S1 IgG titers and their neutralization capacity against the Wuhan strain as well as Alpha and Delta. Overall, our data suggest that even 7 month after the clearance of COVID-19 many patients possess a protective layer of immunity, indicated by the persistence of Spike-specific memory B cells and by the presence of neutralizing antibodies against the Alpha and Delta variants. However, lack of neutralizing antibodies against the Beta, Gamma and Omicron variants even during the peak response is of major concern as this indicates viral evasion of the humoral immune response.

The SARS-unique domain (SUD) of SARS coronavirus contains two macrodomains that bind G-quadruplexes.

Since the outbreak of severe acute respiratory syndrome (SARS) in 2003, the three-dimensional structures of several of the replicase/transcriptase components of SARS coronavirus (SARS-CoV), the non-structural proteins (Nsps), have been determined. However, within the large Nsp3 (1922 amino-acid residues), the structure and function of the so-called SARS-unique domain (SUD) have remained elusive. SUD occurs only in SARS-CoV and the highly related viruses found in certain bats, but is absent from all other coronaviruses. Therefore, it has been speculated that it may be involved in the extreme pathogenicity of SARS-CoV, compared to other coronaviruses, most of which cause only mild infections in humans. In order to help elucidate the function of the SUD, we have determined crystal structures of fragment 389-652 ("SUD(core)") of Nsp3, which comprises 264 of the 338 residues of the domain. Both the monoclinic and triclinic crystal forms (2.2 and 2.8 A resolution, respectively) revealed that SUD(core) forms a homodimer. Each monomer consists of two subdomains, SUD-N and SUD-M, with a macrodomain fold similar to the SARS-CoV X-domain. However, in contrast to the latter, SUD fails to bind ADP-ribose, as determined by zone-interference gel electrophoresis. Instead, the entire SUD(core) as well as its individual subdomains interact with oligonucleotides known to form G-quadruplexes. This includes oligodeoxy- as well as oligoribonucleotides. Mutations of selected lysine residues on the surface of the SUD-N subdomain lead to reduction of G-quadruplex binding, whereas mutations in the SUD-M subdomain abolish it. As there is no evidence for Nsp3 entering the nucleus of the host cell, the SARS-CoV genomic RNA or host-cell mRNA containing long G-stretches may be targets of SUD. The SARS-CoV genome is devoid of G-stretches longer than 5-6 nucleotides, but more extended G-stretches are found in the 3'-nontranslated regions of mRNAs coding for certain host-cell proteins involved in apoptosis or signal transduction, and have been shown to bind to SUD in vitro. Therefore, SUD may be involved in controlling the host cell's response to the viral infection. Possible interference with poly(ADP-ribose) polymerase-like domains is also discussed.

The macromolecular complex of ICP and falcipain-2 from Plasmodium: preparation, crystallization and preliminary X-ray diffraction analysis.

The malaria parasite Plasmodium depends on the tight control of cysteine-protease activity throughout its life cycle. Recently, the characterization of a new class of potent inhibitors of cysteine proteases (ICPs) secreted by Plasmodium has been reported. Here, the recombinant production, purification and crystallization of the inhibitory C-terminal domain of ICP from P. berghei in complex with the P. falciparum haemoglobinase falcipain-2 is described. The 1:1 complex was crystallized in space group P4(3), with unit-cell parameters a = b = 71.15, c = 120.09 Å. A complete diffraction data set was collected to a resolution of 2.6 Å.

ppGpp analogues inhibit synthetase activity of Rel proteins from Gram-negative and Gram-positive bacteria.

A prominent feature of the stringent response is the accumulation of two unusual phosphorylated derivatives of GTP and GDP (pppGpp: 5'-triphosphate-3'-diphosphate, and ppGpp: 5'-3'-bis-diphosphate), collectively called (p)ppGpp, within a few seconds after the onset of amino-acid starvation. The synthesis of these 'alarmone' compounds is catalyzed by RelA homologues. Other features of the stringent response include inhibition of stable RNA synthesis and modulation of transcription, replication, and translation. (p)ppGpp accumulation is important for virulence induction, differentiation and antibiotic resistance. We have synthesized a group of (p)ppGpp analogues and tested them as competitive inhibitors of Rel proteins in vitro. 2'-Deoxyguanosine-3'-5'-di(methylene bisphosphonate) [compound (10)] was found as an inhibitor that reduces ppGpp formation in both Gram-negative and Gram-positive bacteria. In silico docking together with competitive inhibition analysis suggests that compound (10) inhibits activity of Rel proteins by competing with GTP/GDP for its binding site. As Rel proteins are completely absent in mammalians, this appears to be a very attractive approach for the development of novel antibacterial agents.

Association of body surface scanner-based abdominal volume with parameters of the Metabolic Syndrome and comparison with manually measured waist circumference.

To investigate abdominal volume determined by a new body scanner algorithm as anthropometric marker for Metabolic Syndrome (MetS) and its parameters compared to manually measured waist circumference (WC), we performed body scans in 411 participants (38% men, 20-81 years). WC and triglyceride, HDL-cholesterol, and fasting glucose concentrations, and blood pressure were assessed as MetS parameters. We used Spearman correlations and linear regression to investigate associations and goodness-of-fit (R², BIC) of abdominal volume and WC with MetS parameters, and logistic regression to analyse the discriminative power of WC and abdominal volume to assess likelihoods of MetS components and MetS. Correlations with triglyceride, HDL-cholesterol, and glucose concentration were slightly stronger for abdominal volume (r; 0.32, -0.32, and 0.34, respectively) than for WC (0.28, -0.28, and 0.29, respectively). Explained variances in MetS parameters were slightly higher and goodness-of-fit slightly better for abdominal volume than for WC, but differences were small. Exemplarily, glucose levels were 0.28 mmol/L higher (R² = 0.25; BIC = 945.5) per 1-SD higher  WC, and 0.35 mmol/L higher (R² = 0.28; BIC = 929.1) per 1-SD higher abdominal volume. The discriminative power to estimate MetS components was similar for WC and abdominal volume. Our data show that abdominal volume allows metabolic characterization comparable to established WC.

Crystallization and preliminary X-ray analysis of glutathione transferases from cyanobacteria.

Glutathione S-transferases (GSTs) are a group of multifunctional enzymes that are found in animals, plants and microorganisms. Their primary function is to remove toxins derived from exogenous sources or the products of metabolism from the cell. Mammalian GSTs have been extensively studied, in contrast to bacterial GSTs which have received relatively scant attention. A new class of GSTs called Chi has recently been identified in cyanobacteria. Chi GSTs exhibit a high glutathionylation activity towards isothiocyanates, compounds that are normally found in plants. Here, the crystallization of two GSTs are presented: TeGST produced by Thermosynechococcus elongates BP-1 and SeGST from Synechococcus elongates PCC 6301. Both enzymes formed crystals that diffracted to high resolution and appeared to be suitable for further X-ray diffraction studies. The structures of these GSTs may shed further light on the evolution of GST catalytic activity and in particular why these enzymes possess catalytic activity towards plant antimicrobial compounds.

Crystallization and preliminary X-ray diffraction analysis of the ternary human GM-CSF receptor complex.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a haemopoietic growth factor that acts though a ternary receptor signalling complex containing specific alpha (GMRalpha) and common beta (betac) receptor subunits. Human GM-CSF is encoded by the gene csf2, while the genes for GMRalpha and betac are csf2ra and csf2rb, respectively. Crystals of the ternary ectodomain complex comprising GM-CSF and the soluble extracellular regions of both the GMRalpha subunit and either betac or its glutamine-substitution mutant N346Q were obtained using the hanging-drop vapour-diffusion method. The best diffracting crystals of the ternary complex were obtained using the N346Q mutation of the betac subunit. These crystals grew using polyethylene glycol 3350 with a high concentration of proline, belonged to space group P6(3)22 and diffracted to 3.3 A resolution.

Chemogenomic Profiling of Human and Microbial FK506-Binding Proteins.

FK506-binding proteins (FKBPs) are evolutionarily conserved proteins that display peptidyl-prolyl isomerase activities and act as coreceptors for immunosuppressants. Microbial macrophage-infectivity-potentiator (Mip)-type FKBPs can enhance infectivity. However, developing druglike ligands for FKBPs or Mips has proven difficult, and many FKBPs and Mips still lack biologically useful ligands. To explore the scope and potential of C5-substituted [4.3.1]-aza-bicyclic sulfonamides as a broadly applicable class of FKBP inhibitors, we developed a new synthesis method for the bicyclic core scaffold and used it to prepare an FKBP- and Mip-focused library. This allowed us to perform a systematic structure-activity-relationship analysis across key human FKBPs and microbial Mips, yielding highly improved inhibitors for all the FKBPs studied. A cocrystal structure confirmed the molecular-binding mode of the core structure and explained the affinity gained as a result of the preferred substituents. The best FKBP and Mip ligands showed promising antimalarial, antileginonellal, and antichlamydial properties in cellular models of infectivity, suggesting that substituted [4.3.1]-aza-bicyclic sulfonamides could be a novel class of anti-infectives.

Crystal structure of full length topoisomerase I from Thermotoga maritima.

DNA topoisomerases are a family of enzymes altering the topology of DNA by concerted breakage and rejoining of the phosphodiester backbone of DNA. Bacterial and archeal type IA topoisomerases, including topoisomerase I, topoisomerase III, and reverse gyrase, are crucial in regulation of DNA supercoiling and maintenance of genetic stability. The crystal structure of full length topoisomerase I from Thermotoga maritima was determined at 1.7A resolution and represents an intact and fully active bacterial topoisomerase I. It reveals the torus-like structure of the conserved transesterification core domain comprising domains I-IV and a tightly associated C-terminal zinc ribbon domain (domain V) packing against domain IV of the core domain. The previously established zinc-independence of the functional activity of T.maritima topoisomerase I is further supported by its crystal structure as no zinc ion is bound to domain V. However, the structural integrity is preserved by the formation of two disulfide bridges between the four Zn-binding cysteine residues. A functional role of domain V in DNA binding and recognition is suggested and discussed in the light of the structure and previous biochemical findings. In addition, implications for bacterial topoisomerases I are provided.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of chloride intracellular channel 2 (CLIC2).

The chloride intracellular channel (CLIC) family of proteins are unusual in that they can exist in either an integral membrane-channel form or a soluble form. Here, the expression, purification, crystallization and preliminary diffraction analysis of CLIC2, one of the least-studied members of this family, are reported. Human CLIC2 was crystallized in two different forms, both in the presence of reduced glutathione and both of which diffracted to better than 1.9 A resolution. Crystal form A displayed P2(1)2(1)2(1) symmetry, with unit-cell parameters a = 44.0, b = 74.7, c = 79.8 A. Crystal form B displayed P2(1) symmetry, with unit-cell parameters a = 36.0, b = 66.9, c = 44.1 A. Structure determination will shed more light on the structure and function of this enigmatic family of proteins.

Crystal structure of Zika virus NS2B-NS3 protease in complex with a boronate inhibitor.

The ongoing Zika virus (ZIKV) outbreak is linked to severe neurological disorders. ZIKV relies on its NS2B/NS3 protease for polyprotein processing; hence, this enzyme is an attractive drug target. The 2.7 angstrom; crystal structure of ZIKV protease in complex with a peptidomimetic boronic acid inhibitor reveals a cyclic diester between the boronic acid and glycerol. The P2 4-aminomethylphenylalanine moiety of the inhibitor forms a salt-bridge with the nonconserved Asp(83) of NS2B; ion-pairing between Asp(83) and the P2 residue of the substrate likely accounts for the enzyme's high catalytic efficiency. The unusual dimer of the ZIKV protease:inhibitor complex seen in the crystal may provide a model for assemblies formed at high local concentrations of protease at the endoplasmatic reticulum membrane, the site of polyprotein processing.

Structures of DegQ from Legionella pneumophila Define Distinct ON and OFF States.

HtrA (high-temperature requirement A) family proteins play important roles in protein-quality control processes in the bacterial periplasm. A common feature of all members of this family is their modular organization comprising a chymotrypsin-like protease domain and at least one PDZ (postsynaptic density of 95 kDa, disks large homolog 1 and zonula occludens 1) domain. All characterized HtrA proteins assemble into complex oligomers consisting of typically 3-24 monomers, which allow a tight regulation of proteolytic activity. Here, we provide evidence that the assembly of proteolytically active, higher-order complexes of DegQ from Legionella pneumophila is triggered by the binding of substrate-derived peptides. Crystal structures of inactive 3-mers and active 12-mers of DegQ reveal molecular details of elements of a conserved allosteric activation cascade that defines distinct protease ON and OFF states. Results from DegQ(Lp) variants harboring structure-based amino acid substitutions indicate that peptide binding to the PDZ1 domain is critical for proteolytic activity but not for the formation of higher-order oligomers. Combining structural, mutagenesis and biochemical data, we show that, in contrast to the proteolytic activity, the chaperone function of DegQ is not affected by the state of the activation cascade.

Production, crystallization and X-ray diffraction analysis of the protease CT441 from Chlamydia trachomatis.

The prokaryotic obligate intracellular pathogen Chlamydia trachomatis is the most prevalent cause of preventable blindness, affecting approximately six million people worldwide. In addition, C. trachomatis is the most commonly reported sexually transmitted pathogen in Europe and the US, causing pelvic inflammation, ectopic pregnancy and infertility. As in other intracellular pathogens, proteases play crucial roles during most stages of the complex life cycle of Chlamydia. CT441 is a chlamydial protease that has been reported to interfere with oestrogen signalling of the host cell. Here, the recombinant production, purification and crystallization of an inactive variant of CT441, designated CT441° (active-site Ser455 replaced by Ala), are described. CT441° was crystallized in space group P22121, with unit-cell parameters a = 86.7, b = 184.0, c = 209.6 Å. A complete diffraction data set was collected to a resolution of 2.95 Å.