Structural basis for oligosaccharide recognition by Pyrococcus furiosus maltodextrin-binding protein

A maltodextrin-binding protein from Pyrococcus furiosus (PfuMBP) has been overproduced in Escherichia coli, purified, and crystallized. The crystal structure of the protein bound to an oligosaccharide ligand was determined to 1.85 A resolution. The fold of PfuMBP is very similar to that of the orthologous MBP from E. coli (EcoMBP), despite the moderate level of sequence identity between the two proteins (27 % identity, 46 % similarity). PfuMBP is extremely resistant to heat and chemical denaturation, which may be attributed to a number of factors, such as a tightly packed hydrophobic core, clusters of isoleucine residues, salt-bridges, and the presence of proline residues in key positions. Surprisingly, an attempt to crystallize the complex of PfuMBP with maltose resulted in a structure that contained maltotriose in the ligand-binding site. The structure of the complex suggests that there is a considerable energy gain upon binding of maltotriose in comparison to maltose. Moreover, isothermal titration calorimetry experiments demonstrated that the binding of maltotriose to the protein is exothermic and tight, whereas no thermal effect was observed upon addition of maltose at three temperatures. Therefore, PfuMBP evidently is designed to bind oligosaccharides composed of three or more glucopyranose units.     

Structural basis of BLyS receptor recognition

B lymphocyte stimulator (BLyS), a member of the tumor necrosis factor (TNF) superfamily, is a cytokine that induces B-cell proliferation and immunoglobulin secretion. We have determined the three-dimensional structure of BLyS to 2.0 A resolution and identified receptor recognition segments using limited proteolysis coupled with mass spectrometry. Similar to other structurally determined TNF-like ligands, the BLyS monomer is a beta-sandwich and oligomerizes to form a homotrimer. The receptor-binding region in BLyS is a deeper, more pronounced groove than in other cytokines. The conserved elements on the 'floor' of this groove allow for cytokine recognition of several structurally related receptors, whereas variations on the 'walls' and outer rims of the groove confer receptor specificity.     

Conformational Changes Leading to T7 DNA Delivery upon Interaction with the Bacterial Receptor

The majority of bacteriophages protect their genetic material by packaging the nucleic acid in concentric layers to an almost crystalline concentration inside protein shells (capsid). This highly condensed genome also has to be efficiently injected into the host bacterium in a process named ejection. Most phages use a specialized complex (often a tail) to deliver the genome without disrupting cell integrity. Bacteriophage T7 belongs to the Podoviridae family and has a short, non-contractile tail formed by a tubular structure surrounded by fibers. Here we characterize the kinetics and structure of bacteriophage T7 DNA delivery process. We show that T7 recognizes lipopolysaccharides (LPS) from Escherichia coli rough strains through the fibers. Rough LPS acts as the main phage receptor and drives DNA ejection in vitro. The structural characterization of the phage tail after ejection using cryo-electron microscopy (cryo-EM) and single particle reconstruction methods revealed the major conformational changes needed for DNA delivery at low resolution. Interaction with the receptor causes fiber tilting and opening of the internal tail channel by untwisting the nozzle domain, allowing release of DNA and probably of the internal head proteins.     

Structural basis for hormone recognition by the Human CRFR2{alpha} G protein-coupled receptor

The mammalian corticotropin releasing factor (CRF)/urocortin (Ucn) peptide hormones include four structurally similar peptides, CRF, Ucn1, Ucn2, and Ucn3, that regulate stress responses, metabolism, and cardiovascular function by activating either of two related class B G protein-coupled receptors, CRFR1 and CRFR2. CRF and Ucn1 activate both receptors, whereas Ucn2 and Ucn3 are CRFR2-selective. The molecular basis for selectivity is unclear. Here, we show that the purified N-terminal extracellular domains (ECDs) of human CRFR1 and the CRFR2α isoform are sufficient to discriminate the peptides, and we present three crystal structures of the CRFR2α ECD bound to each of the Ucn peptides. The CRFR2α ECD forms the same fold observed for the CRFR1 and mouse CRFR2β ECDs but contains a unique N-terminal α-helix formed by its pseudo signal peptide. The CRFR2α ECD peptide-binding site architecture is similar to that of CRFR1, and binding of the α-helical Ucn peptides closely resembles CRF binding to CRFR1. Comparing the electrostatic surface potentials of the ECDs suggests a charge compatibility mechanism for ligand discrimination involving a single amino acid difference in the receptors (CRFR1 Glu104/CRFR2α Pro-100) at a site proximate to peptide residue 35 (Arg in CRF/Ucn1, Ala in Ucn2/3). CRFR1 Glu-104 acts as a selectivity filter preventing Ucn2/3 binding because the nonpolar Ala-35 is incompatible with the negatively charged Glu-104. The structures explain the mechanisms of ligand recognition and discrimination and provide a molecular template for the rational design of therapeutic agents selectively targeting these receptors.     

Structural basis for oligosaccharide recognition of misfolded glycoproteins by OS-9 in ER-associated degradation

Misfolded glycoproteins are translocated from endoplasmic reticulum (ER) into the cytosol for proteasome-mediated degradation. A mannose-6-phosphate receptor homology (MRH) domain is commonly identified in a variety of proteins and, in the case of OS-9 and XTP3-B, is involved in glycoprotein ER-associated degradation (ERAD). Trimming of outermost α1,2-linked mannose on C-arm of high-mannose-type glycan and binding of processed α1,6-linked mannosyl residues by the MRH domain are critical steps in guiding misfolded glycoproteins to enter ERAD. Here we report the crystal structure of a human OS-9 MRH domain (OS-9(MRH)) complexed with α3,α6-mannopentaose. The OS-9(MRH) has a flattened β-barrel structure with a characteristic P-type lectin fold and possesses distinctive double tryptophan residues in the oligosaccharide-binding site. Our crystallographic result in conjunction with nuclear magnetic resonance (NMR) spectroscopic and biochemical results provides structural insights into the mechanism whereby OS-9 specifically recognizes Manα1,6Manα1,6Man residues on the processed C-arm through the continuous double tryptophan (WW) motif.     

Terminal N-linked galactose is the primary receptor for adeno-associated virus 9

Sialylated glycans serve as cell surface attachment factors for a broad range of pathogens. We report an atypical example, where desialylation increases cell surface binding and infectivity of adeno-associated virus (AAV) serotype 9, a human parvovirus isolate. Enzymatic removal of sialic acid, but not heparan sulfate or chondroitin sulfate, increased AAV9 transduction regardless of cell type. Viral binding and transduction assays on mutant Chinese hamster ovary (CHO) cell lines defective in various stages of glycan chain synthesis revealed a potential role for core glycan residues under sialic acid in AAV9 transduction. Treatment with chemical inhibitors of glycosylation and competitive inhibition studies with different lectins suggest that N-linked glycans with terminal galactosyl residues facilitate cell surface binding and transduction by AAV9. In corollary, resialylation of galactosylated glycans on the sialic acid-deficient CHO Lec2 cell line with different sialyltransferases partially blocked AAV9 transduction. Quantitative analysis of AAV9 binding to parental, sialidase-treated or sialic acid-deficient mutant CHO cells revealed a 3-15-fold increase in relative binding potential of AAV9 particles upon desialylation. Finally, pretreatment of well differentiated human airway epithelial cultures and intranasal instillation of recombinant sialidase in murine airways enhanced transduction efficiency of AAV9 by >1 order of magnitude. Taken together, the studies described herein provide a molecular basis for low infectivity of AAV9 in vitro and a biochemical strategy to enhance gene transfer by AAV9 vectors in general.     

Structural Characterization of the Bacteriophage T7 Tail Machinery

Most bacterial viruses need a specialized machinery, called “tail,” to inject their genomes inside the bacterial cytoplasm without disrupting the cellular integrity. Bacteriophage T7 is a well characterized member of the Podoviridae family infecting Escherichia coli, and it has a short noncontractile tail that assembles sequentially on the viral head after DNA packaging. The T7 tail is a complex of around 2.7 MDa composed of at least four proteins as follows: the connector (gene product 8, gp8), the tail tubular proteins gp11 and gp12, and the fibers (gp17). Using cryo-electron microscopy and single particle image reconstruction techniques, we have determined the precise topology of the tail proteins by comparing the structure of the T7 tail extracted from viruses and a complex formed by recombinant gp8, gp11, and gp12 proteins. Furthermore, the order of assembly of the structural components within the complex was deduced from interaction assays with cloned and purified tail proteins. The existence of common folds among similar tail proteins allowed us to obtain pseudo-atomic threaded models of gp8 (connector) and gp11 (gatekeeper) proteins, which were docked into the corresponding cryo-EM volumes of the tail complex. This pseudo-atomic model of the connector-gatekeeper interaction revealed the existence of a common molecular architecture among viruses belonging to the three tailed bacteriophage families, strongly suggesting that a common molecular mechanism has been favored during evolution to coordinate the transition between DNA packaging and tail assembly.     

Structural basis of M3 muscarinic receptor dimer/oligomer formation

Class A G protein-coupled receptors (GPCRs) are known to form dimers and/or oligomeric arrays in vitro and in vivo. These complexes are thought to play important roles in modulating class A GPCR function. Many studies suggest that residues located on the "outer" (lipid-facing) surface of the transmembrane (TM) receptor core are critically involved in the formation of class A receptor dimers (oligomers). However, no clear consensus has emerged regarding the identity of the TM helices or TM subsegments involved in this process. To shed light on this issue, we have used the M(3) muscarinic acetylcholine receptor (M3R), a prototypic class A GPCR, as a model system. Using a comprehensive and unbiased approach, we subjected all outward-facing residues (70 amino acids total) of the TM helical bundle (TM1-7) of the M3R to systematic alanine substitution mutagenesis. We then characterized the resulting mutant receptors in radioligand binding and functional studies and determined their ability to form dimers (oligomers) in bioluminescence resonance energy transfer saturation assays. We found that M3R/M3R interactions are not dependent on the presence of one specific structural motif but involve the outer surfaces of multiple TM subsegments (TM1-5 and -7) located within the central and endofacial portions of the TM receptor core. Moreover, we demonstrated that the outward-facing surfaces of most TM helices play critical roles in proper receptor folding and/or function. Guided by the bioluminescence resonance energy transfer data, molecular modeling studies suggested the existence of multiple dimeric/oligomeric M3R arrangements, which may exist in a dynamic equilibrium. Given the high structural homology found among all class A GPCRs, our results should be of considerable general relevance.     

Base of the Measles Virus Fusion Trimer Head Receives the Signal That Triggers Membrane Fusion

The measles virus (MV) fusion (F) protein trimer executes membrane fusion after receiving a signal elicited by receptor binding to the hemagglutinin (H) tetramer. Where and how this signal is received is understood neither for MV nor for other paramyxoviruses. Because only the prefusion structure of the parainfluenza virus 5 (PIV5) F-trimer is available, to study signal receipt by the MV F-trimer, we generated and energy-refined a homology model. We used two approaches to predict surface residues of the model interacting with other proteins. Both approaches measured interface propensity values for patches of residues. The second approach identified, in addition, individual residues based on the conservation of physical chemical properties among F-proteins. Altogether, about 50 candidate interactive residues were identified. Through iterative cycles of mutagenesis and functional analysis, we characterized six residues that are required specifically for signal transmission; their mutation interferes with fusion, although still allowing efficient F-protein processing and cell surface transport. One residue is located adjacent to the fusion peptide, four line a cavity in the base of the F-trimer head, while the sixth residue is located near this cavity. Hydrophobic interactions in the cavity sustain the fusion process and contacts with H. The cavity is flanked by two different subunits of the F-trimer. Tetrameric H-stalks may be lodged in apposed cavities of two F-trimers. Because these insights are based on a PIV5 homology model, the signal receipt mechanism may be conserved among paramyxoviruses.     

The energetic basis underpinning T-cell receptor recognition of a super-bulged peptide bound to a major histocompatibility complex class I molecule

Although the major histocompatibility complex class I (MHC-I) molecules typically bind short peptide (p) fragments (8-10 amino acids in length), longer, "bulged" peptides are often be presented by MHC-I. Such bulged pMHC-I complexes represent challenges for T-cell receptor (TCR) ligation, although the general principles underscoring the interaction between TCRs and bulged pMHC-I complexes are unclear. To address this, we have explored the energetic basis of how an immunodominant TCR (termed SB27) binds to a 13-amino acid viral peptide (LPEPLPQGQLTAY) complexed to human leukocyte antigen (HLA) B*3508. Using the crystal structure of the SB27 TCR-HLA B*3508(LPEP) complex as a guide, we undertook a comprehensive alanine-scanning mutagenesis approach at the TCR-pMHC-I interface and examined the effect of the mutations by biophysical (affinity measurements) and cellular approaches (tetramer staining). Although the structural footprint on HLA B*3508 was small, the energetic footprint was even smaller in that only two HLA B*3508 residues were critical for the TCR interaction. Instead, the energetic basis of this TCR-pMHC-I interaction was attributed to peptide-mediated interactions in which the complementarity determining region 3α and germline-encoded complementarity determining region 1β loops of the SB27 TCR played the principal role. Our findings highlight the peptide-centricity of TCR ligation toward a bulged pMHC-I complex.     

Structural basis for promoter DNA recognition by the response regulator OmpR

OmpR, a response regulator of the EnvZ/OmpR two-component system (TCS), controls the reciprocal regulation of two porin proteins, OmpF and OmpC, in bacteria. During signal transduction, OmpR (OmpR-FL) undergoes phosphorylation at its conserved Asp residue in the N-terminal receiver domain (OmpRn) and recognizes the promoter DNA from its C-terminal DNA-binding domain (OmpRc) to elicit an adaptive response. Apart from that, OmpR regulates many genes in Escherichia coli and is important for virulence in several pathogens. However, the molecular mechanism of the regulation and the structural basis of OmpR–DNA binding is still not fully clear. In this study, we presented the crystal structure of OmpRc in complex with the F1 region of the ompF promoter DNA from E. coli. Our structural analysis suggested that OmpRc binds to its cognate DNA as a homodimer, only in a head-to-tail orientation. Also, the OmpRc apo-form showed a unique domain-swapped crystal structure under different crystallization conditions. Biophysical experimental data, such as NMR, fluorescent polarization and thermal stability, showed that inactive OmpR-FL (unphosphorylated) could bind to promoter DNA with a weaker binding affinity as compared with active OmpR-FL (phosphorylated) or OmpRc, and also confirmed that phosphorylation may only enhance DNA binding. Furthermore, the dimerization interfaces in the OmpRc–DNA complex structure identified in this study provide an opportunity to understand the regulatory role of OmpR and explore the potential for this “druggable” target.     

Structural basis for substrate recognition and dissociation by human transportin 1

Transportin 1 (Trn1) is a transport receptor that transports substrates from the cytoplasm to the nucleus through nuclear pore complexes by recognizing nuclear localization signals (NLSs). Here we describe four crystal structures of human Trn1 in a substrate-free form as well as in the complex with three NLSs (hnRNP D, JKTBP, and TAP, respectively). Our data have revealed that (1) Trn1 has two sites for binding NLSs, one with high affinity (site A) and one with low affinity (site B), and NLS interaction at site B controls overall binding affinity for Trn1; (2) Trn1 recognizes the NLSs at site A followed by conformational change at site B to interact with the NLSs; and (3) a long flexible loop, characteristic of Trn1, interacts with site B, thereby displacing transport substrate in the nucleus. These studies provide deep understanding of substrate recognition and dissociation by Trn1 in import pathways.     

Seminal plasma accelerates semen-derived enhancer of viral infection (SEVI) fibril formation by the prostatic acid phosphatase (PAP248-286) peptide

Amyloid fibrils contained in semen, known as SEVI, or semen-derived enhancer of viral infection, have been shown to increase the infectivity of HIV dramatically. However, previous work with these fibrils has suggested that extensive time and nonphysiologic levels of agitation are necessary to induce amyloid formation from the precursor peptide (a proteolytic cleavage product of prostatic acid phosphatase, PAP(248-286)). Here, we show that fibril formation by PAP(248-286) is accelerated dramatically in the presence of seminal plasma (SP) and that agitation is not required for fibrillization in this setting. Analysis of the effects of specific SP components on fibril formation by PAP(248-286) revealed that this effect is primarily due to the anionic buffer components of SP (notably inorganic phosphate and sodium bicarbonate). Divalent cations present in SP had little effect on the kinetics of fibril formation, but physiologic levels of Zn(2+) strongly protected SEVI fibrils from degradation by seminal proteases. Taken together, these data suggest that in the in vivo environment, PAP(248-286) is likely to form fibrils efficiently, thus providing an explanation for the presence of SEVI in human semen.     

Structural basis of carbohydrate recognition by calreticulin

The calnexin cycle is a process by which glycosylated proteins are subjected to folding cycles in the endoplasmic reticulum lumen via binding to the membrane protein calnexin (CNX) or to its soluble homolog calreticulin (CRT). CNX and CRT specifically recognize monoglucosylated Glc₁Man₉GlcNAc₂ glycans, but the structural determinants underlying this specificity are unknown. Here, we report a 1.95-Å crystal structure of the CRT lectin domain in complex with the tetrasaccharide α-Glc-(1→3)-α-Man-(1→2)-α-Man-(1→2)-Man. The tetrasaccharide binds to a long channel on CRT formed by a concave β-sheet. All four sugar moieties are engaged in the protein binding via an extensive network of hydrogen bonds and hydrophobic contacts. The structure explains the requirement for glucose at the nonreducing end of the carbohydrate; the oxygen O₂ of glucose perfectly fits to a pocket formed by CRT side chains while forming direct hydrogen bonds with the carbonyl of Gly¹²⁴ and the side chain of Lys¹¹¹. The structure also explains a requirement for the Cys¹⁰⁵–Cys¹³⁷ disulfide bond in CRT/CNX for efficient carbohydrate binding. The Cys¹⁰⁵–Cys¹³⁷ disulfide bond is involved in intimate contacts with the third and fourth sugar moieties of the Glc₁Man₃ tetrasaccharide. Finally, the structure rationalizes previous mutagenesis of CRT and lays a structural groundwork for future studies of the role of CNX/CRT in diverse biological pathways.     

Structural basis for the specific recognition of IL-18 by its alpha receptor

Interleukin 18 (IL-18), a member of the IL-1 family of cytokines, is an important regulator of innate and acquired immune responses. It signals through its ligand-binding primary receptor IL-18Rα and accessory receptor IL-18Rβ. Here we report the crystal structure of IL-18 with the ectodomain of IL-18Rα, which reveals the structural basis for their specific recognition. It confirms that surface charge complementarity determines the ligand-binding specificity of primary receptors in the IL-1 receptor family. We suggest that IL-18 signaling complex adopts an architecture similar to other agonistic cytokines and propose a general ligand-receptor assembly and activation model for the IL-1 family.     

Structural basis of double-stranded RNA recognition by the RIG-I like receptor MDA5

RIG-I, MDA5 and LGP2 are cytosolic pattern recognition receptors detecting single-stranded or double-stranded RNA in virally infected cells. The activation of RIG-I or MDA5 stimulates the secretion of type I interferons that play key roles in antiviral immune responses. The C-terminal domains (CTD) of RIG-I and LGP2 are responsible for RNA binding; however, it is not clear how MDA5 binds RNA. To understand the structural basis of dsRNA recognition by MDA5, we have determined the 1.45 Å resolution structure of the C-terminal domain of human MDA5. The structure revealed a highly conserved fold similar to the structures of RIG-I and LGP2 CTDs. NMR titration of MDA5 CTD with dsRNA demonstrated that a positively charged surface is involved in dsRNA binding. Mutagenesis and RNA binding studies showed that electrostatic interactions play primary roles in dsRNA recognition by MDA5. Like RIG-I and LGP2, MDA5 CTD preferentially binds dsRNA with blunt ends, but does not associate with dsRNA with either 5′ or 3′ overhangs. Molecular modeling of MDA5 CTD/dsRNA complex suggests that MDA5 CTD may recognize the first turn of blunt-ended dsRNA in a similar manner as LGP2.     

Structural basis of presequence recognition by the mitochondrial protein import receptor Tom20

Most mitochondrial proteins are synthesized in the cytosol as precursor proteins with a cleavable N-terminal presequence and are imported into mitochondria. We report here the NMR structure of a general import receptor, rat Tom20, in a complex with a presequence peptide derived from rat aldehyde dehydrogenase. The cytosolic domain of Tom20 forms an all alpha-helical structure with a groove to accommodate the presequence peptide. The bound presequence forms an amphiphilic helical structure with hydrophobic leucines aligned on one side to interact with a hydrophobic patch in the Tom20 groove. Although the positive charges of the presequence are essential for import ability, presequence binding to Tom20 is mediated mainly by hydrophobic rather than ionic interactions.     

Structural basis of pre-mRNA recognition by the human cleavage factor Im complex

The cleavage factor I(m) (CF I(m)), consists of a 25 kDa subunit (CF I(m)25) and one of three larger subunits (CF I(m)59, CF I(m)68, CF I(m)72), and is an essential protein complex for pre-mRNA 3'-end cleavage and polyadenylation. It recognizes the upstream sequence of the poly(A) site in a sequence-dependent manner. Here we report the crystal structure of human CF I(m), comprising CF I(m)25 and the RNA recognition motif domain of CF I(m)68 (CF I(m)68RRM), and the crystal structure of the CF I(m)-RNA complex. These structures show that two CF I(m)68RRM molecules bind to the CF I(m)25 dimer via a novel RRM-protein interaction mode forming a heterotetramer. The RNA-bound structure shows that two UGUAA RNA sequences, with anti-parallel orientation, bind to one CF I(m)25-CF I(m)68RRM heterotetramer, providing structural basis for the mechanism by which CF I(m) binds two UGUAA elements within one molecule of pre-mRNA simultaneously. Point mutation and kinetic analyses demonstrate that CF I(m)68RRM can bind the immediately flanking upstream region of the UGUAA element, and CF I(m)68RRM binding significantly increases the RNA-binding affinity of the complex, suggesting that CF I(m)68 makes an essential contribution to pre-mRNA binding.     

Structural basis of substrate recognition in human nicotinamide N-methyltransferase

Nicotinamide N-methyltransferase (NNMT) catalyzes the N-methylation of nicotinamide, pyridines, and other analogues using S-adenosyl-l-methionine as donor. NNMT plays a significant role in the regulation of metabolic pathways and is expressed at markedly high levels in several kinds of cancers, presenting it as a potential molecular target for cancer therapy. We have determined the crystal structure of human NNMT as a ternary complex bound to both the demethylated donor S-adenosyl-l-homocysteine and the acceptor substrate nicotinamide, to 2.7 ? resolution. These studies reveal the structural basis for nicotinamide binding and highlight several residues in the active site which may play roles in nicotinamide recognition and NNMT catalysis. The functional importance of these residues was probed by mutagenesis. Of three residues near the nicotinamide's amide group, substitution of S201 and S213 had no effect on enzyme activity while replacement of D197 dramatically decreased activity. Substitutions of Y20, whose side chain hydroxyl interacts with both the nicotinamide aromatic ring and AdoHcy carboxylate, also compromised activity. Enzyme kinetics analysis revealed k(cat)/K(m) decreases of 2-3 orders of magnitude for the D197A and Y20A mutants, confirming the functional importance of these active site residues. The mutants exhibited substantially increased K(m) for both NCA and AdoMet and modestly decreased k(cat). MD simulations revealed long-range conformational effects which provide an explanation for the large increase in K(m)(AdoMet) for the D197A mutant, which interacts directly only with nicotinamide in the ternary complex crystal structure.