Structural basis for peptidoglycan binding by peptidoglycan recognition proteins

Peptidoglycan (PGN) recognition proteins (PGRPs) are pattern-recognition receptors of the innate immune system that bind and, in some cases, hydrolyze bacterial PGNs. We determined the crystal structure, at 2.30-A resolution, of the C-terminal PGN-binding domain of human PGRP-Ialpha in complex with a muramyl tripeptide representing the core of lysine-type PGNs from Gram-positive bacteria. The peptide stem of the ligand is buried at the deep end of a long binding groove, with N-acetylmuramic acid situated in the middle of the groove, whose shallow end can accommodate a linked N-acetylglucosamine. Although most interactions are with the peptide, the glycan moiety also seems to be essential for specific recognition by PGRPs. Conservation of key PGN-contacting residues shows that all PGRPs employ this basic PGN-binding mode. The structure pinpoints variable residues that likely mediate discrimination between lysine- and diaminopimelic acid-type PGNs. We also propose a mechanism for PGN hydrolysis by Zn(2+)-containing PGRPs.     

Structural basis for the recognition and cleavage of abasic DNA in Neisseria meningitidis

Base excision repair (BER) is a highly conserved DNA repair pathway throughout all kingdoms from bacteria to humans. Whereas several enzymes are required to complete the multistep repair process of damaged bases, apurinic-apyrimidic (AP) endonucleases play an essential role in enabling the repair process by recognizing intermediary abasic sites cleaving the phosphodiester backbone 5′ to the abasic site. Despite extensive study, there is no structure of a bacterial AP endonuclease bound to substrate DNA. Furthermore, the structural mechanism for AP-site cleavage is incomplete. Here we report a detailed structural and biochemical study of the AP endonuclease from Neisseria meningitidis that has allowed us to capture structural intermediates providing more complete snapshots of the catalytic mechanism. Our data reveal subtle differences in AP-site recognition and kinetics between the human and bacterial enzymes that may reflect different evolutionary pressures.     

Structural basis for the sequence-specific recognition of human ISG15 by the NS1 protein of influenza B virus

Interferon-induced ISG15 conjugation plays an important antiviral role against several viruses, including influenza viruses. The NS1 protein of influenza B virus (NS1B) specifically binds only human and nonhuman primate ISG15s and inhibits their conjugation. To elucidate the structural basis for the sequence-specific recognition of human ISG15, we determined the crystal structure of the complex formed between human ISG15 and the N-terminal region of NS1B (NS1B-NTR). The NS1B-NTR homodimer interacts with two ISG15 molecules in the crystal and also in solution. The two ISG15-binding sites on the NS1B-NTR dimer are composed of residues from both chains, namely residues in the RNA-binding domain (RBD) from one chain, and residues in the linker between the RBD and the effector domain from the other chain. The primary contact region of NS1B-NTR on ISG15 is composed of residues at the junction of the N-terminal ubiquitin-like (Ubl) domain and the short linker region between the two Ubl domains, explaining why the sequence of the short linker in human and nonhuman primate ISG15s is essential for the species-specific binding of these ISG15s. In addition, the crystal structure identifies NS1B-NTR binding sites in the N-terminal Ubl domain of ISG15, and shows that there are essentially no contacts with the C-terminal Ubl domain of ISG15. Consequently, NS1B-NTR binding to ISG15 would not occlude access of the C-terminal Ubl domain of ISG15 to its conjugating enzymes. Nonetheless, transfection assays show that NS1B-NTR binding of ISG15 is responsible for the inhibition of interferon-induced ISG15 conjugation in cells.     

Structural basis for substrate specificity in the Escherichia coli maltose transport system

ATP-binding cassette (ABC) transporters are molecular pumps that harness the chemical energy of ATP hydrolysis to translocate solutes across the membrane. The substrates transported by different ABC transporters are diverse, ranging from small ions to large proteins. Although crystal structures of several ABC transporters are available, a structural basis for substrate recognition is still lacking. For the Escherichia coli maltose transport system, the selectivity of sugar binding to maltose-binding protein (MBP), the periplasmic binding protein, does not fully account for the selectivity of sugar transport. To obtain a molecular understanding of this observation, we determined the crystal structures of the transporter complex MBP-MalFGK₂ bound with large malto-oligosaccharide in two different conformational states. In the pretranslocation structure, we found that the transmembrane subunit MalG forms two hydrogen bonds with malto-oligosaccharide at the reducing end. In the outward-facing conformation, the transmembrane subunit MalF binds three glucosyl units from the nonreducing end of the sugar. These structural features explain why modified malto-oligosaccharides are not transported by MalFGK₂ despite their high binding affinity to MBP. They also show that in the transport cycle, substrate is channeled from MBP into the transmembrane pathway with a polarity such that both MBP and MalFGK₂ contribute to the overall substrate selectivity of the system.The ATP-binding cassette (ABC) transporter family contains more than 2,000 members sharing a common architecture of two transmembrane domains (TMDs) that form the translocation pathway and two cytoplasmic nucleotide-binding domains (NBDs) that hydrolyze ATP (1). Importers found in prokaryotes require additional soluble proteins that bind substrates with high affinity and deliver them to the TMDs. Some ABC transporters recognize only a single substrate, whereas others are more promiscuous. For example, ABC transporters that secrete toxins, hydrolytic enzymes, and antibiotic peptides are dedicated to one specific substrate (2), but in contrast, the multidrug transporter P-glycoprotein interacts with more than 200 chemically diverse compounds (3). MRP1, ABCG2, and TAP also have broad substrate spectra (2).Regardless of substrate specificity, the ATPase activity of ABC transporters is regulated by the presence of substrates. Thus, substrate binding must generate a signal that enables ATP hydrolysis. Understanding how ABC transporters interact with their substrates has been a major challenge in the field.A controversial issue in the ABC transporter field is whether the transmembrane components contain a well-defined substrate-binding site. It has been suggested that for binding protein-dependent ABC transporters, substrate specificity is defined exclusively by the binding protein, which interacts with the substrate with high affinity. The transmembrane components act as a nonspecific pore for substrate to diffuse through the membrane (4). However, for the Escherichia coli maltose transporter, it has been well established that the selectivity of sugar binding to the maltose-binding protein (MBP) does not fully account for the selectivity of sugar transport. For example, cyclic maltodextrins, maltodextrins containing more than seven glucosyl units, and maltose analogs with a modified reducing end are not transported despite their high-affinity binding to MBP (5, 6). Further evidence for selectivity through the ABC transporter MalFGK₂ itself comes from mutant transporters that function independently of MBP. In the absence of MBP, these mutants constitutively hydrolyze ATP and specifically transport maltodextrins (7, 8).In this study, we determined the crystal structures of the maltose transport complex MBP-MalFGK₂ bound with large maltodextrin in two conformational states. The determination of these structures, along with previous studies of maltoporin and MBP, allow us to define how overall substrate specificity is achieved for the maltose transport system.     

Structural basis for the multiple interactions of the MyD88 TIR domain in TLR4 signaling

Myeloid differentiating factor 88 (MyD88) and MyD88 adaptor-like (Mal) are adaptor molecules critically involved in the Toll-like receptor (TLR) 4 signaling pathway. While Mal has been proposed to serve as a membrane-sorting adaptor, MyD88 mediates signal transduction from activated TLR4 to downstream components. The Toll/Interleukin-1 receptor (TIR) domain of MyD88 is responsible for sorting and signaling via direct or indirect TIR−TIR interactions between Mal and TLR4. However, the molecular mechanisms involved in multiple interactions of the TIR domain remain unclear. The present study describes the solution structure of the MyD88 TIR domain. Reporter gene assays revealed that 3 discrete surface sites in the TIR domain of MyD88 are important for TLR4 signaling. Two of these sites were shown to mediate direct binding to the TIR domain of Mal. Interestingly, Mal-TIR, but not MyD88-TIR, directly binds to the cytosolic TIR domain of TLR4. These observations suggested that the heteromeric assembly of TIR domains of the receptor and adaptors constitutes the initial step of TLR4 intracellular signal transduction.     

Structural basis for complement factor I control and its disease-associated sequence polymorphisms

The complement system is a key component of innate and adaptive immune responses. Complement regulation is critical for prevention and control of disease. We have determined the crystal structure of the complement regulatory enzyme human factor I (fI). FI is in a proteolytically inactive form, demonstrating that it circulates in a zymogen-like state despite being fully processed to the mature sequence. Mapping of functional data from mutants of fI onto the structure suggests that this inactive form is maintained by the noncatalytic heavy-chain allosterically modulating activity of the light chain. Once the ternary complex of fI, a cofactor and a substrate is formed, the allosteric inhibition is released, and fI is oriented for cleavage. In addition to explaining how circulating fI is limited to cleaving only C3b/C4b, our model explains the molecular basis of disease-associated polymorphisms in fI and its cofactors.     

Structural basis for GTP-induced dimerization and antiviral function of guanylate-binding proteins

Guanylate-binding proteins (GBPs) form a family of dynamin-related large GTPases which mediate important innate immune functions. They were proposed to form oligomers upon GTP binding/hydrolysis, but the molecular mechanisms remain elusive. Here, we present crystal structures of C-terminally truncated human GBP5 (hGBP5_1–486), comprising the large GTPase (LG) and middle (MD) domains, in both its nucleotide-free monomeric and nucleotide-bound dimeric states, together with nucleotide-free full-length human GBP2. Upon GTP-loading, hGBP5_1–486 forms a closed face-to-face dimer. The MD of hGBP5 undergoes a drastic movement relative to its LG domain and forms extensive interactions with the LG domain and MD of the pairing molecule. Disrupting the MD interface (for hGBP5) or mutating the hinge region (for hGBP2/5) impairs their ability to inhibit HIV-1. Our results point to a GTP-induced dimerization mode that is likely conserved among all GBP members and provide insights into the molecular determinants of their antiviral function.

Guanylate binding proteins (GBPs) are a family of interferon (IFN)-inducible guanosine triphosphatases (GTPases) that play important roles in innate immunity against diverse intracellular pathogens (1). Many GBPs show activities against bacterial and protozoan pathogens, such as Toxoplasma gondii, Chlamydia trachomatis, Legionella, and Mycobacterium tuberculosis (2). Some of them also have antiviral functions (3). Recently, human GBP5 (hGBP5) was found to restrict HIV-1 by interfering with the processing and incorporation of the viral envelope glycoprotein (Env) (4). A follow-up study revealed that hGBP5 and its paralogue hGBP2 suppress the activity of the virus-dependency factor furin, thereby inhibiting the proteolytic processing of the immature Env precursor gp160 into mature gp120 and gp41 required for virion infectivity (5). Furin is critical for proteolytic cleavage of many viral envelope proteins (6). In support of a key role in innate antiviral immunity, hGBP2 and hGBP5 also restrict other furin-dependent viruses, such as measles, Zika, and highly pathogenic avian influenza A viruses (5).GBPs belong to the dynamin superfamily of large GTPases (7). These are characterized by an N-terminal large GTPase domain (LG domain) and one or more stalk domains (8), usually involved in oligomerization. The stalk domain of GBPs, which is also called C-terminal helical domain (CTHD), comprises the middle domain (MD) and GTPase effector domain (GED). It was proposed that GBPs undergo conformational changes and/or oligomerization upon GTP binding and hydrolysis (9), which may be important for their innate immune functions. Furthermore, GBP1, GBP2, and GBP5 are isoprenylated, and their membrane-binding abilities are modulated by the nucleotide state (10).Despite the importance of this protein family in innate immunity and decades of research, their oligomerization mechanisms remain elusive due to limited structural data (11). The crystal structure of full-length human GBP1 (hGBP1^FL) was determined in its monomeric state (12). The crystal structure of the LG domain alone showed that it is able to form a dimer upon GTP binding (13). Based on these structures, a model of hGBP1^FL in the nucleotide-bound dimeric state was proposed, where the stalk domains protrude to the opposite direction, resulting in an “open” conformation (13). However, the accuracy of this model remains to be tested. Hence, the structures of full-length GBPs in their oligomeric state are in high demand to reveal the detailed molecular mechanisms of GBPs during innate immune responses.Here, we report the crystal structures of hGBP5_1–486 in both its nucleotide-free monomeric state and nucleotide-bound dimeric state, as well as full-length, nucleotide-free human GBP2 (hGBP2^FL). The structures of hGBP5_1–486 and hGBP2^FL are similar to that of hGBP1^FL in the absence of nucleotide. Upon nucleotide binding, however, the stalk domain of hGBP5 undergoes a drastic movement relative to the dimerized LG domain, resulting in a “closed” conformation entirely different from the previously proposed model. Two MD form a hydrophobic interface. Disrupting this interface or mutating the hinge region connecting LG domain and MD, reduces the anti–HIV-1 activity of hGBP2/5, suggesting a crucial role of the closed conformation in their antiviral function. Although the immune functions of the GBP family members are diverse and require specific signals, this dimerization mode is probably shared by all members of the family as revealed by small-angle X-ray scattering (SAXS). On these grounds, we propose a GTP-induced dimerization mechanism of GBPs which lays the foundation to understand the molecular bases of this important innate immune protein family.     

Structural basis for gating mechanisms of a eukaryotic P-glycoprotein homolog

P-glycoprotein is an ATP-binding cassette multidrug transporter that actively transports chemically diverse substrates across the lipid bilayer. The precise molecular mechanism underlying transport is not fully understood. Here, we present crystal structures of a eukaryotic P-glycoprotein homolog, CmABCB1 from Cyanidioschyzon merolae, in two forms: unbound at 2.6-Å resolution and bound to a unique allosteric inhibitor at 2.4-Å resolution. The inhibitor clamps the transmembrane helices from the outside, fixing the CmABCB1 structure in an inward-open conformation similar to the unbound structure, confirming that an outward-opening motion is required for ATP hydrolysis cycle. These structures, along with site-directed mutagenesis and transporter activity measurements, reveal the detailed architecture of the transporter, including a gate that opens to extracellular side and two gates that open to intramembranous region and the cytosolic side. We propose that the motion of the nucleotide-binding domain drives those gating apparatuses via two short intracellular helices, IH1 and IH2, and two transmembrane helices, TM2 and TM5.Multidrug transporters of the ATP-binding cassette (ABC) superfamily, such as P-glycoprotein (P-gp; MDR1; ABCB1), MRP1 (ABCC1), and ABCG2 (BCRP), transport a large number of structurally unrelated compounds with molecular weights ranging up to several thousand Daltons (1, 2). These transporters not only play important roles in normal physiology by protecting tissues from various toxic xenobiotics and endogenous metabolites but also contribute to multidrug resistance (MDR) in tumors, a major obstacle to effective chemotherapeutic treatment (1, 3–7). Their functional forms consist of a minimum of four core domains: two transmembrane domains (TMDs) that create the translocation pathway for substrates and two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP to power the transport process (8, 9). These four domains can exist either as two separate polypeptides (half-size) or fused together in a single large polypeptide with an internal duplication (full-size). The crystal structures of mouse and nematode P-gps, as well as their bacterial homologs (10–14), have been determined, and they have provided important insights into the relationships between protein structure and the functional and biochemical characteristics of P-gp. However, the detailed architecture of the TMD machinery and the gating mechanism during the transition between the inward- and outward-open states are poorly understood.Here, we report the structures of a eukaryotic P-gp homolog, unlocked (at 2.6-Å resolution) and locked allosterically with a tailor-made peptide at 2.4-Å resolution. Although CmABCB1 is not a full-length ABC transporter but a half-sized ABC transporter adopting a homodimeric architecture, CmABCB1 showed quite similar functional properties to those of human P-gp (hP-gp). Based on these structures, we propose mechanisms by which the intramembranous entrance gate can take up various substrates from the inner leaflet of the membrane bilayer. Although the entrance and exit gates are assembled from distinct transmembrane helices, and their gating motions are in opposite directions, the mechanisms powered by the dimerization motions of the NBDs enable one gate to close while the other gate simultaneously opens with the aid of two short intracellular helices and two transmembrane helices, which act as a lever arm. This mechanism is totally different from that of solute carrier (SLC) transporters (15–19) and ABC importers (20), in which the intra- and extracellular gating apparatuses are constructed from the same transmembrane helices. Furthermore, a part of the intramembranous entrance gate has another function gating the pathway from the inside of the transporter to the cytosol. The mode of action of the novel inhibitor, which disables the diverging outward motions of the TM helices by clamping them from the outside of the transporter, supports our proposed gating mechanism.     

Structural basis for the evolution of vancomycin resistance D,D-peptidases

Vancomycin resistance in Gram-positive bacteria is due to production of cell-wall precursors ending in d-Ala-d-Lac or d-Ala-d-Ser, to which vancomycin exhibits low binding affinities, and to the elimination of the high-affinity precursors ending in d-Ala-d-Ala. Depletion of the susceptible high-affinity precursors is catalyzed by the zinc-dependent d,d-peptidases VanX and VanY acting on dipeptide (d-Ala-d-Ala) or pentapeptide (UDP-MurNac-l-Ala-d-Glu-l-Lys-d-Ala-d-Ala), respectively. Some of the vancomycin resistance operons encode VanXY d,d-carboxypeptidase, which hydrolyzes both di- and pentapeptide. The molecular basis for the diverse specificity of Van d,d-peptidases remains unknown. We present the crystal structures of VanXY_C and VanXY_G in apo and transition state analog-bound forms and of VanXY_C in complex with the d-Ala-d-Ala substrate and d-Ala product. Structural and biochemical analysis identified the molecular determinants of VanXY dual specificity. VanXY residues 110–115 form a mobile cap over the catalytic site, whose flexibility is involved in the switch between di- and pentapeptide hydrolysis. Structure-based alignment of the Van d,d-peptidases showed that VanY enzymes lack this element, which promotes binding of the penta- rather than that of the dipeptide. The structures also highlight the molecular basis for selection of d-Ala–ending precursors over the modified resistance targets. These results illustrate the remarkable adaptability of the d,d-peptidase fold in response to antibiotic pressure via evolution of specific structural elements that confer hydrolytic activity against vancomycin-susceptible peptidoglycan precursors.The emergence of high-level resistance to vancomycin, a last resort antibiotic against Gram-positive bacteria, in Enterococcus spp. and its spread to methicillin-resistant Staphylococcus aureus is a serious threat to public health (1). Vancomycin acts by binding to the d-alanyl-d-alanine moiety of the uncross-linked N-acetyl-muramyl-l-Ala-d-γ-Glu-l-Lys-d-Ala-d-Ala (pentapeptide[d-Ala]) peptidoglycan precursor blocking the extracellular steps in peptidoglycan synthesis. Resistance is mediated by nine types of operons that replace the d-Ala-d-Ala terminus of peptidoglycan precursors with d-Ala-d-lactate (VanA, -B, -D, and -M types) or d-Ala-d-serine (VanC, -E, -G, -L, and -N types), to which vancomycin exhibits lower binding affinities (2).A critical step in vancomycin resistance involves depletion of d-Ala–terminating precursors to prevent interaction of vancomycin with its target. This step is facilitated by the d,d-dipeptidase VanX and the d,d-pentapeptidase VanY, which hydrolyze, respectively, d-Ala-d-Ala and the C-terminal d-Ala residue from pentapeptide[d-Ala] (3–6). In the d-Ala-d-Ser form of resistance, a single VanXY enzyme evolved to mediate both d,d-dipeptidase and d,d-pentapeptidase activities (7, 8). Thus, Van d,d-peptidases demonstrate variation in peptidoglycan substrate selectivity that correlates with the specific resistance mechanism. As part of the d-Ala-d-Lac type resistance mechanism, the VanX enzyme shows 10⁵-fold higher catalytic efficiency against d-Ala-d-Ala compared with d-Ala-d-Lac substrates, thus facilitating accumulation of the depsipeptide. However, VanX retains significant activity against d-Ala-d-Ser dipeptide (9). Appropriate to its role in resistance, the VanY enzyme shows carboxypeptidase activity against pentapeptide[d-Ala] but lacks activity against d,d-dipeptide substrates (5, 10). The VanXY_C enzyme is selective against resistant dipeptide and pentapeptide peptidoglycan substrates ending in d-Ser (8). Finally, the VanXY_G enzyme, first assigned as a dual substrate active “XY” enzyme by sequence similarity (11), was later shown to lack d,d-pentapeptidase activity typical for bona fide VanXY enzymes (12). The molecular determinants responsible for such diversity of substrate specificity among Van d,d-peptidases are unknown, limiting the understanding of their evolution and hampering the development of inhibitors that could be used in combination with glycopeptides.With the exception of VanY_D, which is a penicillin-binding protein (13), all VanX, VanY, and VanXY d,d-peptidases are zinc-dependent enzymes classified into the metallopeptidase clan MD, family M15, according to the MEROPS database (14). Within clan MD, multiple families of enzymes, including M15 representatives, are involved in bacterial cell wall metabolism (15). Three members of this family have been structurally characterized: zinc d-Ala-d-Ala carboxypeptidase from Streptomyces albus (16) (subfamily M15A), bacteriophage l-Ala-d-Glu peptidase PLY500 (17) (subfamily M15C), and VanX (subfamily M15D) from Enterococcus faecium (18). These enzymes display a common core tertiary fold built on a central antiparallel β-sheet arrayed with multiple α-helices on either face of the β-sheet. The fold contains two consensus motifs that form the active site, His-X(6)-Asp and Glu-X(2)-His, with a zinc ion coordinated by the histidine and aspartate residues. In addition, the structure of VanX revealed a small and constricted active site that explains its specificity toward the d-Ala-d-Ala substrate (18). The VanY and VanXY enzymes belonging to subfamily M15B remain structurally uncharacterized.Given the evolution toward dual substrate specificity of VanXY enzymes, their importance in resistance, and their sequence and functional diversity, we undertook their detailed structural and functional characterization. We determined the crystal structures of VanXY_C and VanXY_G and performed extensive mutagenesis analysis. Our data led to the identification and characterization of the molecular features responsible for their substrate specificities and demonstrated an exceptional diversification and plasticity within their common metallopeptidase fold in response to drug selective pressure.     

Structural basis of the C1q/C1s interaction and its central role in assembly of the C1 complex of complement activation

Complement component C1, the complex that initiates the classical pathway of complement activation, is a 790-kDa assembly formed from the target-recognition subcomponent C1q and the modular proteases C1r and C1s. The proteases are elongated tetramers that become more compact when they bind to the collagen-like domains of C1q. Here, we describe a series of structures that reveal how the subcomponents associate to form C1. A complex between C1s and a collagen-like peptide containing the C1r/C1s-binding motif of C1q shows that the collagen binds to a shallow groove via a critical lysine side chain that contacts Ca²⁺-coordinating residues. The data explain the Ca²⁺-dependent binding mechanism, which is conserved in C1r and also in mannan-binding lectin-associated serine proteases, the serine proteases of the lectin pathway activation complexes. In an accompanying structure, C1s forms a compact ring-shaped tetramer featuring a unique head-to-tail interaction at its center that replicates the likely arrangement of C1r/C1s polypeptides in the C1 complex. Additional structures reveal how C1s polypeptides are positioned to enable activation by C1r and interaction with the substrate C4 inside the cage-like assembly formed by the collagenous stems of C1q. Together with previously determined structures of C1r fragments, the results reported here provide a structural basis for understanding the early steps of complement activation via the classical pathway.     

Structures of the flax-rust effector AvrM reveal insights into the molecular basis of plant-cell entry and effector-triggered immunity

Fungal and oomycete pathogens cause some of the most devastating diseases in crop plants, and facilitate infection by delivering a large number of effector molecules into the plant cell. AvrM is a secreted effector protein from flax rust (Melampsora lini) that can internalize into plant cells in the absence of the pathogen, binds to phosphoinositides (PIPs), and is recognized directly by the resistance protein M in flax (Linum usitatissimum), resulting in effector-triggered immunity. We determined the crystal structures of two naturally occurring variants of AvrM, AvrM-A and avrM, and both reveal an L-shaped fold consisting of a tandem duplicated four-helix motif, which displays similarity to the WY domain core in oomycete effectors. In the crystals, both AvrM variants form a dimer with an unusual nonglobular shape. Our functional analysis of AvrM reveals that a hydrophobic surface patch conserved between both variants is required for internalization into plant cells, whereas the C-terminal coiled-coil domain mediates interaction with M. AvrM binding to PIPs is dependent on positive surface charges, and mutations that abrogate PIP binding have no significant effect on internalization, suggesting that AvrM binding to PIPs is not essential for transport of AvrM across the plant membrane. The structure of AvrM and the identification of functionally important surface regions advance our understanding of the molecular mechanisms underlying how effectors enter plant cells and how they are detected by the plant immune system.Filamentous eukaryotic microbes such as fungi and oomycetes cause devastating diseases in many economically important crop plants, including rice, corn, wheat, soybean, and potato. During infection, oomycetes and biotrophic fungal pathogens establish a physical interaction with their host through specialized feeding structures, known as haustoria, which facilitate secretion of a vast array of effector proteins to overcome plant defenses and promote host colonization (1–3). The effectors are generally highly divergent even among related species, and generally lack similarity to proteins currently available in the databases, making it difficult to predict biological function from sequence alone. Some of the effectors accumulate in the plant intercellular space (apoplast), whereas others are translocated into the host cell. At present, the host targets and virulence mechanisms of fungal and oomycete effectors are poorly understood. A subset of the plant-translocated effectors called avirulence (Avr) proteins are recognized directly or indirectly and with high specificity by plant disease resistance (R) proteins (4–6). This recognition event leads to activation of effector-triggered immunity (ETI), which usually includes rapid localized host cell death at the site of infection—termed the hypersensitive response (HR)—and results in the plants being immune to pathogen infection.The question of how fungal and oomycete effector proteins are transported across the host-cell membrane is currently a topic of considerable interest and controversy. Delivery of effectors into host cells by eukaryotic pathogens has been demonstrated in several plant pathogen systems (7–12), and, in some cases, fungal and oomycete host-cell translocation can occur in the absence of the pathogen (7, 12–16), suggesting that a host-encoded transport machinery is responsible for internalization of the effectors. Conserved short N-terminal motifs such as RXLR and LXLFLAK have been identified in oomycete-effector protein families (17), and they have been shown to be both necessary and sufficient for plant-cell entry (9, 16, 18). Although the N-terminal regions of some fungal effectors including AvrL567 and AvrM from flax rust are required for plant-cell entry (7), conserved uptake signals have so far not been identified in fungal pathogens. RXLR motifs in oomycete effectors, and RXLR-like motifs in some fungal effectors, have been reported to be required for host-cell translocation and binding to phosphatidylinositol 3-phosphate (PI3P). Based on these findings, Kale et al. (15) proposed that oomycete and fungal effector proteins are translocated into the host cytoplasm after binding to PI3P associated with the external face of the cell, in a process that involves lipid raft-mediated endocytosis. However, this view has recently been challenged by reports showing that the C-terminal effector domain of AVR3a from Phytophthora infestans, and not the N-terminal region harboring the RXLR motif, is required for binding to PI3P (19–21). There is also an ongoing debate in the literature about the significance of the RXLR motifs in host-cell translocation (22, 23).The interaction between flax and the fungal pathogen Melampsora lini (flax rust) has been adopted as a model system for studying plant–fungal rust interactions. Several M. lini effector genes have been identified (AvrL567, AvrM, AvrP123, and AvrP4) and they encode small, secreted proteins that are recognized by TIR-NB-LRR (Toll-interleukin receptor, nucleotide binding, leucine-rich repeat) R proteins inside the plant cell (24–27). The AvrM effector is delivered into host cells during infection, and interacts directly with the M resistance protein (7, 28). AvrM has no significant sequence similarity to proteins of known structure and its internalization mechanism, virulence functions, and host targets are unknown. Six naturally occurring variants (AvrM-A to AvrM-E and avrM) have been identified. AvrM-A to AvrM-D are recognized by M and induce HR, whereas AvrM-E and avrM evade recognition (26). The C-terminal region of AvrM-A (residues 106–343) forms a structured and protease-resistant domain that can dimerize in plants and in solution (28). On transient expression in planta, secreted AvrM internalizes into the plant cell cytosol and deletion studies have revealed that residues 123 to 153 are necessary and sufficient for this internalization (7), whereas the C-terminal region (residues 225–343) is required for M-dependent ETI (28). AvrM-A can also bind to negatively charged phospholipids including PI3P, but the role of PIP binding in host-cell translocation is not clear, and the lipid-binding sites do not overlap with the region identified for AvrM uptake (29).To understand the molecular basis of plant-cell translocation, PIP binding and AvrM:M interaction, we solved crystal structures of the C-terminal domains of two different AvrM variants, AvrM-A and avrM. AvrM-A and avrM share 93% amino acid sequence identity in the C-terminal shared region, and also differ by a 62-aa internal deletion in avrM and a 34-aa deletion at the C terminus of AvrM-A. Both structures reveal a unique L-shaped helical fold and form a dimer with an unusual nonglobular shape. Surprisingly, the structures contain two four-helix repeats that share some similarity to the overall architecture of the WY domain in oomycete effectors (30). Analysis of the structure combined with site-directed mutagenesis reveals that a conserved hydrophobic surface patch is required for pathogen-independent internalization, but that PIP binding is surface charge-dependent and not required for this process. Mapping of AvrM-A deletion mutants demonstrates that M recognizes the C-terminal coiled-coil domain of AvrM-A, and mutational analysis of single polymorphic residues in AvrM-A and avrM suggest that multiple contact points are required for the M:AvrM interaction.     

Structural basis for effectiveness of siderophore-conjugated monocarbams against clinically relevant strains of Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that causes nosocomial infections for which there are limited treatment options. Penicillin-binding protein PBP3, a key therapeutic target, is an essential enzyme responsible for the final steps of peptidoglycan synthesis and is covalently inactivated by β-lactam antibiotics. Here we disclose the first high resolution cocrystal structures of the P. aeruginosa PBP3 with both novel and marketed β-lactams. These structures reveal a conformational rearrangement of Tyr532 and Phe533 and a ligand-induced conformational change of Tyr409 and Arg489. The well-known affinity of the monobactam aztreonam for P. aeruginosa PBP3 is due to a distinct hydrophobic aromatic wall composed of Tyr503, Tyr532, and Phe533 interacting with the gem-dimethyl group. The structure of MC-1, a new siderophore-conjugated monocarbam complexed with PBP3 provides molecular insights for lead optimization. Importantly, we have identified a novel conformation that is distinct to the high-molecular-weight class B PBP subfamily, which is identifiable by common features such as a hydrophobic aromatic wall formed by Tyr503, Tyr532, and Phe533 and the structural flexibility of Tyr409 flanked by two glycine residues. This is also the first example of a siderophore-conjugated triazolone-linked monocarbam complexed with any PBP. Energetic analysis of tightly and loosely held computed hydration sites indicates protein desolvation effects contribute significantly to PBP3 binding, and analysis of hydration site energies allows rank ordering of the second-order acylation rate constants. Taken together, these structural, biochemical, and computational studies provide a molecular basis for recognition of P. aeruginosa PBP3 and open avenues for future design of inhibitors of this class of PBPs.     

Absent in melanoma 2 is required for innate immune recognition of Francisella tularensis

Macrophages respond to cytosolic nucleic acids by activating cysteine protease caspase-1 within a complex called the inflammasome. Subsequent cleavage and secretion of proinflammatory cytokines IL-1β and IL-18 are critical for innate immunity. Here, we show that macrophages from mice lacking absent in melanoma 2 (AIM2) cannot sense cytosolic double-stranded DNA and fail to trigger inflammasome assembly. Caspase-1 activation in response to intracellular pathogen Francisella tularensis also required AIM2. Immunofluorescence microscopy of macrophages infected with F. tularensis revealed striking colocalization of bacterial DNA with endogenous AIM2 and inflammasome adaptor ASC. By contrast, type I IFN (IFN-α and -β) secretion in response to F. tularensis did not require AIM2. IFN-I did, however, boost AIM2-dependent caspase-1 activation by increasing AIM2 protein levels. Thus, inflammasome activation was reduced in infected macrophages lacking either the IFN-I receptor or stimulator of interferon genes (STING). Finally, AIM2-deficient mice displayed increased susceptibility to F. tularensis infection compared with wild-type mice. Their increased bacterial burden in vivo confirmed that AIM2 is essential for an effective innate immune response.     

Structural basis for induced formation of the inflammatory mediator prostaglandin E2

Prostaglandins (PG) are bioactive lipids produced from arachidonic acid via the action of cyclooxygenases and terminal PG synthases. Microsomal prostaglandin E synthase 1 (MPGES1) constitutes an inducible glutathione-dependent integral membrane protein that catalyzes the oxidoreduction of cyclooxygenase derived PGH(2) into PGE(2). MPGES1 has been implicated in a number of human diseases or pathological conditions, such as rheumatoid arthritis, fever, and pain, and is therefore regarded as a primary target for development of novel antiinflammatory drugs. To provide a structural basis for insight in the catalytic mechanism, we determined the structure of MPGES1 in complex with glutathione by electron crystallography from 2D crystals induced in the presence of phospholipids. Together with results from site-directed mutagenesis and activity measurements, we can thereby demonstrate the role of specific amino acid residues. Glutathione is found to bind in a U-shaped conformation at the interface between subunits in the protein trimer. It is exposed to a site facing the lipid bilayer, which forms the specific environment for the oxidoreduction of PGH(2) to PGE(2) after displacement of the cytoplasmic half of the N-terminal transmembrane helix. Hence, insight into the dynamic behavior of MPGES1 and homologous membrane proteins in inflammation and detoxification is provided.     

Structural basis for allosteric cross-talk between the asymmetric nucleotide binding sites of a heterodimeric ABC exporter

ATP binding cassette (ABC) transporters mediate vital transport processes in every living cell. ATP hydrolysis, which fuels transport, displays positive cooperativity in numerous ABC transporters. In particular, heterodimeric ABC exporters exhibit pronounced allosteric coupling between a catalytically impaired degenerate site, where nucleotides bind tightly, and a consensus site, at which ATP is hydrolyzed in every transport cycle. Whereas the functional phenomenon of cooperativity is well described, its structural basis remains poorly understood. Here, we present the apo structure of the heterodimeric ABC exporter TM287/288 and compare it to the previously solved structure with adenosine 5′-(β,γ-imido)triphosphate (AMP-PNP) bound at the degenerate site. In contrast to other ABC exporter structures, the nucleotide binding domains (NBDs) of TM287/288 remain in molecular contact even in the absence of nucleotides, and the arrangement of the transmembrane domains (TMDs) is not influenced by AMP-PNP binding, a notion confirmed by double electron-electron resonance (DEER) measurements. Nucleotide binding at the degenerate site results in structural rearrangements, which are transmitted to the consensus site via two D-loops located at the NBD interface. These loops owe their name from a highly conserved aspartate and are directly connected to the catalytically important Walker B motif. The D-loop at the degenerate site ties the NBDs together even in the absence of nucleotides and substitution of its aspartate by alanine is well-tolerated. By contrast, the D-loop of the consensus site is flexible and the aspartate to alanine mutation and conformational restriction by cross-linking strongly reduces ATP hydrolysis and substrate transport.ABC exporters are found in every organism (1, 2). They minimally consist of four domains and exist as homodimers or heterodimers. Two transmembrane domains (TMDs) span the membrane with a total of 12 transmembrane helices and form the substrate permeation pathway by alternating between inward- and outward-oriented states (Fig. S1A). A pair of nucleotide binding domains (NBDs) is connected to the TMDs via coupling helices and drive conformational cycling of the transporter by binding and hydrolysis of ATP, a process which is linked to NBD dimerization and dissociation (3).In their closed state, the NBDs sandwich two ATP molecules at the dimer interface by composite ATP binding sites involving conserved sequence motifs contributed by both subunits (4, 5). The A-loop and Walker A motif of one NBD and the ABC signature motif of the opposite NBD are involved in nucleotide binding. The Walker B glutamate and the switch-loop histidine constitute a catalytic dyad required for ATP hydrolysis (6, 7). In heterodimeric ABC exporters with asymmetric ATP binding sites, these catalytic residues are noncanonical at the degenerate site and ATP is therefore primarily, if not exclusively, hydrolyzed at the consensus site (8). The Q- and D-loops were associated with interdomain communication (3, 9–11), but their functional role remains poorly understood.Recently, we reported the structure of the heterodimeric ABC exporter TM287/288 from the thermophilic bacterium Thermotoga maritima, which was crystallized in the presence of adenosine 5′-(β,γ-imido)triphosphate (AMP-PNP) and was shown to transport drugs and dyes when expressed in Lactococcus lactis (12). The transporter adopted an inward-facing state with a nucleotide bound exclusively to the degenerate site. In contrast to the inward-oriented structures of MsbA (13), ABCB10 (14), and P-glycoprotein (15–17) in which the NBDs are separated or twisted (18), we found that the NBDs of TM287/288 remain in close contact and do not shift in the NBD dimerization plane (Fig. S1). The current transport mechanism of TM287/288 envisages the binding of a second nucleotide to the consensus site for the transition to the outward-facing NBD-closed state, which subsequently is hydrolyzed to permit resetting of the transporter (12).Here, we present the high-resolution structure of the nucleotide-free state of TM287/288. Despite high ATP concentrations in the cell, this state is transiently adopted during transport; at the consensus site, the hydrolysis product ADP is replaced by ATP in each transport cycle, and at the degenerate site, the bound nucleotide is occasionally exchanged. We show that the asymmetric NBDs of TM287/288 remain in contact even in the absence of nucleotides. By comparing the apo state with the AMP-PNP–bound structure, we unravel the structural basis for allosteric coupling between the ATP binding sites.     

Structural insights into the mechanisms of Mg2+ uptake,transport, and gating by CorA

Despite the importance of Mg²⁺ for numerous cellular activities, the mechanisms underlying its import and homeostasis are poorly understood. The CorA family is ubiquitous and is primarily responsible for Mg²⁺ transport. However, the key questions—such as, the ion selectivity, the transport pathway, and the gating mechanism—have remained unanswered for this protein family. We present a 3.2 Å resolution structure of the archaeal CorA from Methanocaldococcus jannaschii, which is a unique complete structure of a CorA protein and reveals the organization of the selectivity filter, which is composed of the signature motif of this family. The structure reveals that polar residues facing the channel coordinate a partially hydrated Mg²⁺ during the transport. Based on these findings, we propose a unique gating mechanism involving a helical turn upon the binding of Mg²⁺ to the regulatory intracellular binding sites, and thus converting a polar ion passage into a narrow hydrophobic pore. Because the amino acids involved in the uptake, transport, and gating are all conserved within the entire CorA family, we believe this mechanism is general for the whole family including the eukaryotic homologs.     

Structural basis for endosomal trafficking of diverse transmembrane cargos by PX-FERM proteins

Transit of proteins through the endosomal organelle following endocytosis is critical for regulating the homeostasis of cell-surface proteins and controlling signal transduction pathways. However, the mechanisms that control these membrane-transport processes are poorly understood. The Phox-homology (PX) domain-containing proteins sorting nexin (SNX) 17, SNX27, and SNX31 have emerged recently as key regulators of endosomal recycling and bind conserved Asn-Pro-Xaa-Tyr–sorting signals in transmembrane cargos via an atypical band, 4.1/ezrin/radixin/moesin (FERM) domain. Here we present the crystal structure of the SNX17 FERM domain bound to the sorting motif of the P-selectin adhesion protein, revealing both the architecture of the atypical FERM domain and the molecular basis for recognition of these essential sorting sequences. We further show that the PX-FERM proteins share a promiscuous ability to bind a wide array of putative cargo molecules, including receptor tyrosine kinases, and propose a model for their coordinated molecular interactions with membrane, cargo, and regulatory proteins.     

Structural basis of actin recognition and arginine ADP-ribosylation by Clostridium perfringens iota-toxin

The ADP-ribosylating toxins (ADPRTs) produced by pathogenic bacteria modify intracellular protein and affect eukaryotic cell function. Actin-specific ADPRTs (including Clostridium perfringens iota-toxin and Clostridium botulinum C2 toxin) ADP-ribosylate G-actin at Arg-177, leading to disorganization of the cytoskeleton and cell death. Although the structures of many actin-specific ADPRTs are available, the mechanisms underlying actin recognition and selective ADP-ribosylation of Arg-177 remain unknown. Here we report the crystal structure of actin-Ia in complex with the nonhydrolyzable NAD analog betaTAD at 2.8 A resolution. The structure indicates that Ia recognizes actin via five loops around NAD: loop I (Tyr-60-Tyr-62 in the N domain), loop II (active-site loop), loop III, loop IV (PN loop), and loop V (ADP-ribosylating turn-turn loop). We used site-directed mutagenesis to confirm that loop I on the N domain and loop II are essential for the ADP-ribosyltransferase activity. Furthermore, we revealed that Glu-378 on the EXE loop is in close proximity to Arg-177 in actin, and we proposed that the ADP-ribosylation of Arg-177 proceeds by an SN1 reaction via first an oxocarbenium ion intermediate and second a cationic intermediate by alleviating the strained conformation of the first oxocarbenium ion. Our results suggest a common reaction mechanism for ADPRTs. Moreover, the structure might be of use in rational drug design to block toxin-substrate recognition.