X-ray crystal structure of the C4d fragment of human complement component C4

C4 fulfills a vital role in the propagation of the classical and lectin pathways of the complement system. Although there are no reports to date of a C4 functional activity that is mediated solely by the C4d region, evidence clearly points to it having a vital role in a number of the properties of native C4 and its major activation fragment, C4b. Contained within the C4d region are the thioester-forming residues, the four isotype-specific residues controlling the C4A/C4B transacylation preferences, a binding site for nascent C3b important in assembling the classical pathway C5 convertase and determinants for the Chido/Rodgers (Ch/Rg) blood group antigens. In view of its functional importance, we undertook to determine the three-dimensional structure of C4d by X-ray crystallography. Here we report the 2.3A resolution structure of C4Ad, the C4d fragment derived from the human C4A isotype. Although the approximately 30% sequence identity between C4Ad and the corresponding fragment of C3 might be expected to establish a general fold similarity between the two molecules, C4Ad in fact displays a fold that is essentially superimposable on the structure of C3d. By contrast, the electrostatic characteristics of the various faces of the C4Ad molecule show marked differences from the corresponding faces of C3d, likely reflecting the differences in function between C3 and C4. Residues previously predicted to form the major Ch/Rg epitopes were proximately located and accessible on the concave surface of C4Ad. In addition to providing further insights on the current models for the covalent binding reaction, the C4Ad structure allows one to rationalize why C4d is not a ligand for complement receptor 2. Finally the structure allows for the visualization of the face of the molecule containing the binding site for C3b utilized in the assembly of classical pathway C5 convertase.     

Covalent binding properties of the human complement protein C4 and hydrolysis rate of the internal thioester upon activation.

The complement proteins C3 and C4 have an internal thioester. Upon activation on the surface of a target cell, the thioester becomes exposed and reactive to surface-bound amino and hydroxyl groups, thus allowing covalent deposition of C3 and C4 on these targets. The two human C4 isotypes, C4A and C4B, which differ by only four amino acids, have different binding specificities. C4A binds more efficiently than C4B to amino groups, and C4B is more effective than C4A in binding to hydroxyl groups. By site-directed mutagenesis, the four residues in a cDNA clone of C4B were modified. The variants were expressed and their binding properties studied. Variants with a histidine residue at position 1106 showed C4B-like binding properties, and those with aspartic acid, alanine, or asparagine at the same position were C4A-like. These results suggest that the histidine is important in catalyzing the reaction of the thioester with water and other hydroxyl group-containing compounds. When substituted with other amino acids, this reaction is not catalyzed and the thioester becomes apparently more reactive with amino groups. This interpretation also predicts that the stability of the thioester in C4A and C4B, upon activation, will be different. We measured the time course of activation and binding of glycine to C4A and C4B. The lag in the binding curve behind the activation curve for C4A is significantly greater than that for C4B. The hydrolysis rates (k0) of the thioester in the activated proteins were estimated to be 0.068 s-1 (t1/2 of 10.3 s) for C4A and 1.08 s-1 (t1/2 of 0.64 s) for C4B. These results indicate that the difference in hydrolysis rate of the thioester accounts, at least in part, for the difference in the binding properties of C4A and C4B.     

The structure of bovine lysosomal alpha-mannosidase suggests a novel mechanism for low-pH activation

Lysosomal alpha-mannosidase (LAM: EC 3.2.1.24) belongs to the sequence-based glycoside hydrolase family 38 (GH38). Two other mammalian GH38 members, Golgi alpha-mannosidase II (GIIAM) and cytosolic alpha-mannosidase, are expressed in all tissues. In humans, cattle, cat and guinea pig, lack of lysosomal alpha-mannosidase activity causes the autosomal recessive disease alpha-mannosidosis. Here, we describe the three-dimensional structure of bovine lysosomal alpha-mannosidase (bLAM) at 2.7A resolution and confirm the solution state dimer by electron microscopy. We present the first structure of a mammalian GH38 enzyme that offers indications for the signal areas for mannose phosphorylation, suggests a previously undetected mechanism of low-pH activation and provides a template for further biochemical studies of the family 38 glycoside hydrolases as well as lysosomal transport. Furthermore, it provides a basis for understanding the human form of alpha-mannosidosis at the atomic level. The atomic coordinates and structure factors have been deposited in the Protein Data Bank (accession codes 1o7d and r1o7dsf).     

Insights into complement convertase formation based on the structure of the factor B‐cobra venom factor complex

Immune protection by the complement system critically depends on assembly of C3 convertases on the surface of pathogens and altered host cells. These short‐lived protease complexes are formed through pro‐convertases, which for the alternative pathway consist of the complement component C3b and the pro‐enzyme factor B (FB). Here, we present the crystal structure at 2.2‐Å resolution, small‐angle X‐ray scattering and electron microscopy (EM) data of the pro‐convertase formed by human FB and cobra venom factor (CVF), a potent homologue of C3b that generates more stable convertases. FB is loaded onto CVF through its pro‐peptide Ba segment by specific contacts, which explain the specificity for the homologous C3b over the native C3 and inactive products iC3b and C3c. The protease segment Bb binds the carboxy terminus of CVF through the metal‐ion dependent adhesion site of the Von Willebrand factor A‐type domain. A possible dynamic equilibrium between a ‘loading’ and ‘activation’ state of the pro‐convertase may explain the observed difference between the crystal structure of CVFB and the EM structure of C3bB. These insights into formation of convertases provide a basis for further development of complement therapeutics.     

The structure of Rigidoporus lignosus Laccase containing a full complement of copper ions, reveals an asymmetrical arrangement for the T3 copper pair

Laccase is a multicopper blue oxidase that couples the four-electron reduction of oxygen with the oxidation of a broad range of organic substrates, including phenols and arylamines. The enzyme is the object of intense biotechnological research, due to its employment in bioremediation of soils and water as well as in other biotechnological applications. We report here the cDNA and protein sequences, the post-translational modifications, the crystallization and X-ray structure determination of a laccase from the white-rot fungus Rigidoporus lignosus. The amino acid residues sequence deduced from cDNA clearly identified a pre-sequence of 21 residues representing the signal for extra-cellular localization. Mass spectrometry analysis performed on the salvage enzyme, confirmed the deduced sequence and precisely mapped two glycosylation sites at Asn337 and Asn435, determining the nature of the bound glycosidic moieties. The crystal structure was determined at 1.7A resolution from perfectly hemihedrally twinned crystals, by molecular replacement technique. While the overall structure closely resembled those reported for other fungal laccases, the analysis of the T2/T3 trinuclear cluster revealed an unprecedented coordination sphere for the T3 copper pair. No bridging oxygen ligand was present between the two T3 copper ions, which were no longer symmetrically coordinated. The observed structure could represent an intermediate along the process of four-electron reduction of oxygen to water taking place at the trinuclear copper cluster.     

Solution structure of Compstatin,a potent complement inhibitor.

The third component of complement, C3, plays a central role in activation of the classical, alternative, and lectin pathways of complement activation. Recently, we have identified a 13-residue cyclic peptide (named Compstatin) that specifically binds to C3 and inhibits complement activation. To investigate the topology and the contribution of each critical residue to the binding of Compstatin to C3, we have now determined the solution structure using 2D NMR techniques; we have also synthesized substitution analogues and used these to study the structure-function relationships involved. Finally, we have generated an ensemble of a family of solution structures of the peptide with a hybrid distance geometry-restrained simulated-annealing methodology, using distance, dihedral angle, and 3J(NH-Halpha)-coupling constant restraints. The Compstatin structure contained a type I beta-turn comprising the segment Gln5-Asp6-Trp7-Gly8. Preference for packing of the hydrophobic side chains of Val3, Val4, and Trp7 was observed. The generated structure was also analyzed for consistency using NMR parameters such as NOE connectivity patterns, 3J(NH-Halpha)-coupling constants, and chemical shifts. Analysis of Ala substitution analogues suggested that Val3, Gln5, Asp6, Trp7, and Gly8 contribute significantly to the inhibitory activity of the peptide. Substitution of Gly8 caused a 100-fold decrease in inhibitory potency. In contrast, substitution of Val4, His9, His10, and Arg11 resulted in minimal change in the activity. These findings indicate that specific side-chain interactions and the beta-turn are critical for preservation of the conformational stability of Compstatin and they might be significant for maintaining the functional activity of Compstatin.     

Structural requirements for thioester bond formation in human complement component C3. Reassessment of the role of thioester bond integrity on the conformation of C3.

A unique thioester bond, formed between the side chains of neighboring C and Q residues, is present in complement components C3 and C4 and the protease inhibitor alpha 2-macroglobulin. This structure is essential for mediating covalent attachment to target acceptors and also for maintaining these proteins in their native conformation. An examination of the residues in the immediate vicinity of the C and Q reveals a very high degree of sequence similarity among the three proteins which crosses species barriers. The following is the sequence flanking the thioester residues in C3, the highly conserved amino acids being underlined and the the thioester-forming residues being indicated by italics: 1005V-T-P-S-G-C-G-E-Q-N-M-I-G-M-T-P-T1021. Through a site-directed mutagenesis and cDNA expression approach, we have examined the importance of the conserved amino acids in the formation, stability, and function of the thioester bond in C3. The behavior of the mutants fell into three categories. The potential loss in peptide backbone flexibility by the replacement of G1009 by A or S was permissive to thioester formation and function as was replacement of M1015 by the still fairly bulky residue F. In contrast, replacement of M1015 by A resulted in an alpha-chain which was highly unstable toward proteolytic degradation. The third category, which included mutant molecules P1007G, P1020G, E1012Q, and Q1013N, displayed an unusual phenotype in which both the autolytic fragmentation and the hemolytic activity characteristics of thioester-intact molecules were absent. However, like their wildtype counterpart, these molecules retained the ability to be cleaved by C3 convertase (C4b2a), a conformation-dependent property that is normally lost in the conversion of native C3 to thioester-hydrolyzed C3(H2O). Since an identical functional profile was obtained when the thioester was deliberately prevented from forming in the mutant C1010A, we conclude that if a stable thioester fails to form during biosynthesis, at least parts of the mature protein can adopt a more native-like conformation than is the case when the thioester is first formed and then hydrolyzed in the mature protein. In view of these new findings, the interpretation of the previously observed correlation between the loss of thioester integrity and the adoption of a C3b-like conformation must be reassessed.     

Population data and a fourth allele for equine complement component 3 (C3)

The C3 polymorphism of equine serum or plasma revealed by agarose gel electrophoresis can be diagnosed with protein stain following acid protein fixation. In addition to the three alleles previously described (C31, C32, C33), a fourth allele (C34) was found. Population data for 25 domestic breeds and Equus przewalskii are presented.     

The structure of OMCI, a novel lipocalin inhibitor of the complement system

The complement (C) system is a potent innate immune defence system against parasites. We have recently characterised and expressed OmCI, a 16 kDa protein derived from the soft tick Ornithodoros moubata that specifically binds C5, thereby preventing C activation. The structure of recombinant OmCI determined at 1.9 A resolution confirms a lipocalin fold and reveals that the protein binds a fatty acid derivative that we have identified by mass spectrometry as ricinoleic acid. We propose that OmCI could sequester one of the fatty acid-derived inflammatory modulators from the host plasma, thereby interfering with the host inflammatory response to the tick bite. Mapping of sequence differences between OmCI and other tick lipocalins with different functions, combined with biochemical investigations of OmCI activity, supports the hypothesis that OmCI acts by preventing interaction with the C5 convertase, rather than by blocking the C5a cleavage site.     

The structure of the Escherichia coli nucleoside diphosphate kinase reveals a new quaternary architecture for this enzyme family

Nucleoside diphosphate kinase (NDPK) catalyzes the transfer of gamma-phosphate from nucleoside triphosphates to nucleoside diphosphates. The subunit folding and the dimeric basic structural unit are remarkably the same for available structures but, depending on species, dimers self-associate to form hexamers or tetramers. The crystal structure of the Escherichia coli NDPK reveals a new tetrameric quaternary structure for this protein family. The two tetramers differ by the relative orientation of interacting dimers, which face either the convex or the concave side of their central sheet as in either Myxococcus xanthus (type I) or E. coli (type II), respectively. In the type II tetramer, the subunits interact by a new interface harboring a zone called the Kpn loop as in hexamers, but by the opposite face of this loop. The evolutionary conservation of the interface residues indicates that this new quaternary structure seems to be the most frequent assembly mode in bacterial tetrameric NDP kinases.     

The structure of Atg4B–LC3 complex reveals the mechanism of LC3 processing and delipidation during autophagy

Atg8 is conjugated to phosphatidylethanolamine (PE) by ubiquitin‐like conjugation reactions. Atg8 has at least two functions in autophagy: membrane biogenesis and target recognition. Regulation of PE conjugation and deconjugation of Atg8 is crucial for these functions in which Atg4 has a critical function by both processing Atg8 precursors and deconjugating Atg8–PE. Here, we report the crystal structures of catalytically inert human Atg4B (HsAtg4B) in complex with processed and unprocessed forms of LC3, a mammalian orthologue of yeast Atg8. On LC3 binding, the regulatory loop and the N‐terminal tail of HsAtg4B undergo large conformational changes. The regulatory loop masking the entrance of the active site of free HsAtg4B is lifted by LC3 Phe119, so that a groove is formed along which the LC3 tail enters the active site. At the same time, the N‐terminal tail masking the exit of the active site of HsAtg4B in the free form is detached from the enzyme core and a large flat surface is exposed, which might enable the enzyme to access the membrane‐bound LC3–PE.     

Crystal structure of Bombyx mori arylphorins reveals a 3:3 heterohexamer with multiple papain cleavage sites

In holometabolous insects, the accumulation and utilization of storage proteins (SPs), including arylphorins and methionine‐rich proteins, are critical for the insect metamorphosis. SPs function as amino acids reserves, which are synthesized in fat body, secreted into the larval hemolymph and taken up by fat body shortly before pupation. However, the detailed molecular mechanisms of digestion and utilization of SPs during development are largely unknown. Here, we report the crystal structure of Bombyx mori arylphorins at 2.8 Å, which displays a heterohexameric structural arrangement formed by trimerization of dimers comprising two structural similar arylphorins. Our limited proteolysis assay and microarray data strongly suggest that papain‐like proteases are the major players for B. mori arylphorins digestion in vitro and in vivo. Consistent with the biochemical data, dozens of papain cleavage sites are mapped on the surface of the heterohexameric structure of B. mori arylphorins. Hence, our results provide the insightful information to understand the metamorphosis of holometabolous insects at molecular level.     

The structure around the thioester bond in bovine alpha 2-macroglobulin. Possible implications for the conformational stability of the inhibitor on thioester cleavage

The residues contributing to the thioester bonds in bovine alpha 2-macroglobulin were differentially labelled by modification of the Glu moiety with [14C]methylamine and of the Cys moiety with iodo[3H]acetate. The labelled region was identified and analyzed in a tryptic peptide. Two amino acid replacements between human and bovine alpha 2-macroglobulin were found at positions +3 (Val/Ala) and +4 (Leu/Arg) from the Glu moiety of the thioester. Thus, marked differences exist between the human and bovine proteins in side chain size and charge close to the thioester bonds. These differences may explain the greater conformational stability of bovine alpha 2-macroglobulin, compared with that of the human inhibitor, after cleavage of the thioester bonds.     

Structure and function of interleukin-17 family cytokines

The recently identified interleukin-17 (IL-17) cytokines family, which comprises six members in mammals (IL-17A-F), plays essential roles in the host immunity against infectious diseases and chronic inflammatory diseases. The three-dimensional structures containing IL-17A or IL-17F have become available and revealed the unique structural features of IL-17s as well as their receptors. Molecular modeling in this review shows that IL-17s may adopt a “cysteine knot” fold commonly seen in nerve growth factor (NGF) and other neurotrophins. Further modeling analysis unmasks a signature interaction feature of the IL-17F/IL-17RA complex, where a small loop of IL-17RA slots into the deep groove of the interface of IL-17F homodimer. This is quite different from the interaction between the best known four-helix cytokines and their cognate receptors. On the other hand, structure of IL-17A and its monoclonal antibody (CAT-2200) shows that, albeit that the antigenic epitope of IL-17A resides outside of the IL-17A homodimer interface, its physical proximity to the receptor binding groove may explain that antibody blockage would be achieved by interfering with the ligand-receptor interaction. This review is to summarize the advance in understanding the structure and function of IL-17 family cytokines, focusing mainly on IL-17A, IL-17F and IL-17E, in the hope of gaining better knowledge of immunotherapeutic strategies against various inflammatory diseases.     

The solution structure of the complement deregulator FHR5 reveals a compact dimer and provides new insights into CFHR5 nephropathy

The human complement Factor H–related 5 protein (FHR5) antagonizes the main circulating complement regulator Factor H, resulting in the deregulation of complement activation. FHR5 normally contains nine short complement regulator (SCR) domains, but a FHR5 mutant has been identified with a duplicated N-terminal SCR-1/2 domain pair that causes CFHR5 nephropathy. To understand how this duplication causes disease, we characterized the solution structure of native FHR5 by analytical ultracentrifugation and small-angle X-ray scattering. Sedimentation velocity and X-ray scattering indicated that FHR5 was dimeric, with a radius of gyration (R_g) of 5.5 ± 0.2 nm and a maximum protein length of 20 nm for its 18 domains. This result indicated that FHR5 was even more compact than the main regulator Factor H, which showed an overall length of 26–29 nm for its 20 SCR domains. Atomistic modeling for FHR5 generated a library of 250,000 physically realistic trial arrangements of SCR domains for scattering curve fits. Only compact domain structures in this library fit well to the scattering data, and these structures readily accommodated the extra SCR-1/2 domain pair present in CFHR5 nephropathy. This model indicated that mutant FHR5 can form oligomers that possess additional binding sites for C3b in FHR5. We conclude that the deregulation of complement regulation by the FHR5 mutant can be rationalized by the enhanced binding of FHR5 oligomers to C3b deposited on host cell surfaces. Our FHR5 structures thus explained key features of the mechanism and pathology of CFHR5 nephropathy.     

The structure of GFRalpha1 domain 3 reveals new insights into GDNF binding and RET activation

Glial cell line-derived neurotrophic factor (GDNF) binds to the GDNF family co-receptor alpha1 (GFRalpha1) and activates RET receptor tyrosine kinase. GFRalpha1 has a putative domain structure of three homologous cysteine-rich domains, where domains 2 and 3 make up a central domain responsible for GDNF binding. We report here the 1.8 A crystal structure of GFRalpha1 domain 3 showing a new protein fold. It is an all-alpha five-helix bundle with five disulfide bridges. The structure was used to model the homologous domain 2, the other half of the GDNF-binding fragment, and to construct the first structural model of the GDNF-GFRalpha1 interaction. Using site-directed mutagenesis, we identified closely spaced residues, Phe213, Arg224, Arg225 and Ile229, comprising a putative GDNF-binding surface. Mutating each one of them had slightly different effects on GDNF binding and RET phosphorylation. In addition, the R217E mutant bound GDNF equally well in the presence and absence of RET. Arg217 may thus be involved in the allosteric properties of GFRalpha1 or in binding RET.     

Solution structure of the complex formed between human complement C3d and full-length complement receptor type 2

Complement receptor type 2 (CR2, CD21) is a cell surface protein that links the innate and adaptive immune response during the activation of B-cells through its binding to C3d, a cleavage fragment of the major complement component C3. The extracellular portion of CR2 comprises 15 or 16 short complement regulator (SCR) domains in a partially folded-back but flexible structure. Here, the effect of C3d binding to CR2 was determined by analytical ultracentrifugation and X-ray scattering. The sedimentation coefficient of unbound CR2 is 4.03 S in 50 mM NaCl. Because this agrees well with a value of 3.93 S in 137 mM NaCl, the overall CR2 structure is unaffected by change in ionic strength. Unbound C3d exists in monomer-dimer and monomer-trimer equilibria in 50 mM NaCl, but as a monomer only in 137 mM NaCl. In c(s) size-distribution analyses, an equimolar mixture of the CR2-C3d complex in 50 mM NaCl revealed a single peak shifted to 4.52 S when compared to unbound CR2 at 4.03 S to show that the complex had formed. The CR2-C3d complex in 137 mM NaCl showed two peaks at 2.52 S and 4.07 S to show that this had dissociated. Solution structural models for the CR2 SCR-1/2 complex with C3d and CR2 SCR-1/15 were superimposed. These gave an average sedimentation coefficient of 4.57 S for the complex, in good agreement with the observed value of 4.52 S. It is concluded that CR2 does not detectably change conformation when C3d is bound to it. Consistent with previous analyses, its C3d complex is not formed in physiological salt conditions. The implications of these solution results for its immune role are discussed. To our knowledge, this is the first solution structural study of a large multidomain SCR protein CR2 bound to its physiological ligand C3d.     

The crystal structure of Zika virus NS5 reveals conserved drug targets

Zika virus (ZIKV) has emerged as major health concern, as ZIKV infection has been shown to be associated with microcephaly, severe neurological disease and possibly male sterility. As the largest protein component within the ZIKV replication complex, NS5 plays key roles in the life cycle and survival of the virus through its N-terminal methyltransferase (MTase) and C-terminal RNA-dependent RNA polymerase (RdRp) domains. Here, we present the crystal structures of ZIKV NS5 MTase in complex with an RNA cap analogue (^m7GpppA) and the free NS5 RdRp. We have identified the conserved features of ZIKV NS5 MTase and RdRp structures that could lead to development of current antiviral inhibitors being used against flaviviruses, including dengue virus and West Nile virus, to treat ZIKV infection. These results should inform and accelerate the structure-based design of antiviral compounds against ZIKV.