Lachlan W. Casey

Structural Basis for Assembly and Function of a Heterodimeric Plant Immune Receptor

Universal Immune Function Certain pathogen effectors are detected in plants by cytoplasmic receptors. First solving the crystal structures of Arabidopsis receptors, Williams et al. (p. 299; see the Perspective by Nishimura and Dangl) discovered that in the resting state, the structures form a heterodimer that readies the complex for effector binding and keeps the signaling domains from firing too early. Once the pathogen effector binds, the structure of the complex shifts such that the signaling domains can form a homodimer to initiate downstream signaling. Similarities between these plant-pathogen receptors and Toll-like receptors in animals suggest the molecular mechanisms may translate broadly. A heterodimer stands at the ready; a homodimer responds with action. [Also see Perspective by Nishimura and Dangl] Cytoplasmic plant immune receptors recognize specific pathogen effector proteins and initiate effector-triggered immunity. In Arabidopsis, the immune receptors RPS4 and RRS1 are both required to activate defense to three different pathogens. We show that RPS4 and RRS1 physically associate. Crystal structures of the N-terminal Toll–interleukin-1 receptor/resistance (TIR) domains of RPS4 and RRS1, individually and as a heterodimeric complex (respectively at 2.05, 1.75, and 2.65 angstrom resolution), reveal a conserved TIR/TIR interaction interface. We show that TIR domain heterodimerization is required to form a functional RRS1/RPS4 effector recognition complex. The RPS4 TIR domain activates effector-independent defense, which is inhibited by the RRS1 TIR domain through the heterodimerization interface. Thus, RPS4 and RRS1 function as a receptor complex in which the two components play distinct roles in recognition and signaling.

Structure-Informed Design of an Enzymatically Inactive Vaccine Component for Group A Streptococcus

ABSTRACT Streptococcus pyogenes (group A Streptococcus [GAS]) causes ~700 million human infections/year, resulting in >500,000 deaths. There is no commercial GAS vaccine available. The GAS surface protein arginine deiminase (ADI) protects mice against a lethal challenge. ADI is an enzyme that converts arginine to citrulline and ammonia. Administration of a GAS vaccine preparation containing wild-type ADI, a protein with inherent enzymatic activity, may present a safety risk. In an approach intended to maximize the vaccine safety of GAS ADI, X-ray crystallography and structural immunogenic epitope mapping were used to inform vaccine design. This study aimed to knock out ADI enzyme activity without disrupting the three-dimensional structure or the recognition of immunogenic epitopes. We determined the crystal structure of ADI at 2.5 Å resolution and used it to select a number of amino acid residues for mutagenesis to alanine (D166, E220, H275, D277, and C401). Each mutant protein displayed abrogated activity, and three of the mutant proteins (those with the D166A, H275A, and D277A mutations) possessed a secondary structure and oligomerization state equivalent to those of the wild type, produced high-titer antisera, and avoided disruption of B-cell epitopes of ADI. In addition, antisera raised against the D166A and D277A mutant proteins bound to the GAS cell surface. The inactivated D166A and D277A mutant ADIs are ideal for inclusion in a GAS vaccine preparation. There is no human ortholog of ADI, and we confirm that despite limited structural similarity in the active-site region to human peptidyl ADI 4 (PAD4), ADI does not functionally mimic PAD4 and antiserum raised against GAS ADI does not recognize human PAD4. IMPORTANCE We present an example of structural biology informing human vaccine design. We previously showed that the administration of the enzyme arginine deiminase (ADI) to mice protected the mice against infection with multiple GAS serotypes. In this study, we determined the structure of GAS ADI and used this information to improve the vaccine safety of GAS ADI. Catalytically inactive mutant forms of ADI retained structure, recognition by antisera, and immunogenic epitopes, rendering them ideal for inclusion in GAS vaccine preparations. This example of structural biology informing vaccine design may underpin the formulation of a safe and efficacious GAS vaccine. We present an example of structural biology informing human vaccine design. We previously showed that the administration of the enzyme arginine deiminase (ADI) to mice protected the mice against infection with multiple GAS serotypes. In this study, we determined the structure of GAS ADI and used this information to improve the vaccine safety of GAS ADI. Catalytically inactive mutant forms of ADI retained structure, recognition by antisera, and immunogenic epitopes, rendering them ideal for inclusion in GAS vaccine preparations. This example of structural biology informing vaccine design may underpin the formulation of a safe and efficacious GAS vaccine.

The CC domain structure from the wheat stem rust resistance protein Sr33 challenges paradigms for dimerization in plant NLR proteins

Significance Plants and animals use intracellular immunity receptors, known as nucleotide-binding oligomerization domain-like receptors (NLRs), to defend themselves against invading microbes. In this study, we report the solution structure of the N-terminal coiled-coil (CC) domain from the wheat stem rust resistance protein Sr33. Remarkably, this structure differs substantially from the published crystal structure of the equivalent region from the orthologous barley powdery mildew resistance protein MLA10. Using a structural, biophysical, and functional approach, we compare the Sr33 CC domain with other structurally defined NLR CC domains. Collectively, this work redefines our current understanding of the structure and function of plant NLR CC domains, which has significant implications for future studies into this important class of defense receptors. Plants use intracellular immunity receptors, known as nucleotide-binding oligomerization domain-like receptors (NLRs), to recognize specific pathogen effector proteins and induce immune responses. These proteins provide resistance to many of the world’s most destructive plant pathogens, yet we have a limited understanding of the molecular mechanisms that lead to defense signaling. We examined the wheat NLR protein, Sr33, which is responsible for strain-specific resistance to the wheat stem rust pathogen, Puccinia graminis f. sp. tritici. We present the solution structure of a coiled-coil (CC) fragment from Sr33, which adopts a four-helix bundle conformation. Unexpectedly, this structure differs from the published dimeric crystal structure of the equivalent region from the orthologous barley powdery mildew resistance protein, MLA10, but is similar to the structure of the distantly related potato NLR protein, Rx. We demonstrate that these regions are, in fact, largely monomeric and adopt similar folds in solution in all three proteins, suggesting that the CC domains from plant NLRs adopt a conserved fold. However, larger C-terminal fragments of Sr33 and MLA10 can self-associate both in vitro and in planta, and this self-association correlates with their cell death signaling activity. The minimal region of the CC domain required for both cell death signaling and self-association extends to amino acid 142, thus including 22 residues absent from previous biochemical and structural protein studies. These data suggest that self-association of the minimal CC domain is necessary for signaling but is likely to involve a different structural basis than previously suggested by the MLA10 crystallographic dimer.

Mechanism of Bacterial Interference with TLR4 Signaling by Brucella Toll/Interleukin-1 Receptor Domain-containing Protein TcpB

Background: TcpB is a TIR domain-containing protein form Brucella. Results: TcpB interacts with the host Toll-like receptor and adaptors, and its structure reveals a dimer essential for activity. Conclusion: TcpB forms a nonfunctional complex with host molecules, thus suppressing signaling. Significance: The work explains the structural and functional basis of immune suppression by the protein TcpB from a pathogenic bacterium. Upon activation of Toll-like receptors (TLRs), cytoplasmic Toll/interleukin-1 receptor (TIR) domains of the receptors undergo homo- or heterodimerization. This in turn leads to the recruitment of adaptor proteins, activation of transcription factors, and the secretion of pro-inflammatory cytokines. Recent studies have described the TIR domain-containing protein from Brucella melitensis, TcpB (BtpA/Btp1), to be involved in virulence and suppression of host innate immune responses. TcpB interferes with TLR4 and TLR2 signaling pathways by a mechanism that remains controversial. In this study, we show using co-immunoprecipitation analyses that TcpB interacts with MAL, MyD88, and TLR4 but interferes only with the MAL-TLR4 interaction. We present the crystal structure of the TcpB TIR domain, which reveals significant structural differences in the loop regions compared with other TIR domain structures. We demonstrate that TcpB forms a dimer in solution, and the crystal structure reveals the dimerization interface, which we validate by mutagenesis and biophysical studies. Our study advances the understanding of the molecular mechanisms of host immunosuppression by bacterial pathogens.

Multiple functional self-association interfaces in plant TIR domains

Significance Toll/interleukin-1 receptor/resistance protein (TIR) domains are present in plant and animal innate immunity receptors and appear to play a scaffold function in defense signaling. In both systems, self-association of TIR domains is crucial for their function. In plants, the TIR domain is associated with intracellular immunity receptors, known as nucleotide-binding oligomerization domain-like receptors (NLRs). Previous studies from several plant NLRs have identified two distinct interfaces that are required for TIR:TIR dimerization in different NLRs. We show that the two interfaces previously identified are both important for self-association and defense signaling of multiple TIR–NLR proteins. Collectively, this work suggests that there is a common mechanism of TIR domain self-association in signaling across the TIR–NLR class of receptor proteins. The self-association of Toll/interleukin-1 receptor/resistance protein (TIR) domains has been implicated in signaling in plant and animal immunity receptors. Structure-based studies identified different TIR-domain dimerization interfaces required for signaling of the plant nucleotide-binding oligomerization domain-like receptors (NLRs) L6 from flax and disease resistance protein RPS4 from Arabidopsis. Here we show that the crystal structure of the TIR domain from the Arabidopsis NLR suppressor of npr1-1, constitutive 1 (SNC1) contains both an L6-like interface involving helices αD and αE (DE interface) and an RPS4-like interface involving helices αA and αE (AE interface). Mutations in either the AE- or DE-interface region disrupt cell-death signaling activity of SNC1, L6, and RPS4 TIR domains and full-length L6 and RPS4. Self-association of L6 and RPS4 TIR domains is affected by mutations in either region, whereas only AE-interface mutations affect SNC1 TIR-domain self-association. We further show two similar interfaces in the crystal structure of the TIR domain from the Arabidopsis NLR recognition of Peronospora parasitica 1 (RPP1). These data demonstrate that both the AE and DE self-association interfaces are simultaneously required for self-association and cell-death signaling in diverse plant NLRs.

Structure and function of the TIR domain from the grape NLR protein RPV1

The N-terminal Toll/interleukin-1 receptor/resistance protein (TIR) domain has been shown to be both necessary and sufficient for defense signaling in the model plants flax and Arabidopsis. In examples from these organisms, TIR domain self-association is required for signaling function, albeit through distinct interfaces. Here, we investigate these properties in the TIR domain containing resistance protein RPV1 from the wild grapevine Muscadinia rotundifolia. The RPV1 TIR domain, without additional flanking sequence present, is autoactive when transiently expressed in tobacco, demonstrating that the TIR domain alone is capable of cell-death signaling. We determined the crystal structure of the RPV1 TIR domain at 2.3 Å resolution. In the crystals, the RPV1 TIR domain forms a dimer, mediated predominantly through residues in the αA and αE helices (“AE” interface). This interface is shared with the interface discovered in the dimeric complex of the TIR domains from the Arabidopsis RPS4/RRS1 resistance protein pair. We show that surface-exposed residues in the AE interface that mediate the dimer interaction in the crystals are highly conserved among plant TIR domain-containing proteins. While we were unable to demonstrate self-association of the RPV1 TIR domain in solution or using yeast 2-hybrid, mutations of surface-exposed residues in the AE interface prevent the cell-death autoactive phenotype. In addition, mutation of residues known to be important in the cell-death signaling function of the flax L6 TIR domain were also shown to be required for RPV1 TIR domain mediated cell-death. Our data demonstrate that multiple TIR domain surfaces control the cell-death function of the RPV1 TIR domain and we suggest that the conserved AE interface may have a general function in TIR-NLR signaling.

Structural and Biochemical Analysis of a Single Amino-Acid Mutant of WzzBSF That Alters Lipopolysaccharide O-Antigen Chain Length in Shigella flexneri.

Lipopolysaccharide (LPS), a surface polymer of Gram-negative bacteria, helps bacteria survive in different environments and acts as a virulence determinant of host infection. The O-antigen (Oag) component of LPS exhibits a modal chain-length distribution that is controlled by polysaccharide co-polymerases (PCPs). The molecular basis of the regulation of Oag chain-lengths remains unclear, despite extensive mutagenesis and structural studies of PCPs from Escherichia coli and Shigella. Here, we identified a single mutation (A107P) of the Shigella flexneri WzzBSF, by a random mutagenesis approach, that causes a shortened Oag chain-length distribution in bacteria. We determined the crystal structures of the periplasmic domains of wild-type WzzBSF and the A107P mutant. Both structures form a highly similar open trimeric assembly in the crystals, and show a similar tendency to self-associate in solution. Binding studies by bio-layer interferometry reveal cooperative binding of very short (VS)-core-plus-O-antigen polysaccharide (COPS) to the periplasmic domains of both proteins, but with decreased affinity for the A107P mutant. Our studies reveal that subtle and localized structural differences in PCPs can have dramatic effects on LPS chain-length distribution in bacteria, for example by altering the affinity for the substrate, which supports the role of the structure of the growing Oag polymer in this process.

Stability of the Octameric Structure Affects Plasminogen-Binding Capacity of Streptococcal Enolase

Group A Streptococcus (GAS) is a human pathogen that has the potential to cause invasive disease by binding and activating human plasmin(ogen). Streptococcal surface enolase (SEN) is an octameric α-enolase that is localized at the GAS cell surface. In addition to its glycolytic role inside the cell, SEN functions as a receptor for plasmin(ogen) on the bacterial surface, but the understanding of the molecular basis of plasmin(ogen) binding is limited. In this study, we determined the crystal and solution structures of GAS SEN and characterized the increased plasminogen binding by two SEN mutants. The plasminogen binding ability of SENK312A and SENK362A is ~2- and ~3.4-fold greater than for the wild-type protein. A combination of thermal stability assays, native mass spectrometry and X-ray crystallography approaches shows that increased plasminogen binding ability correlates with decreased stability of the octamer. We propose that decreased stability of the octameric structure facilitates the access of plasmin(ogen) to its binding sites, leading to more efficient plasmin(ogen) binding and activation.

Small-Angle X-Ray Scattering for the Discerning Macromolecular Crystallographer

The role of small-angle X-ray scattering (SAXS) in structural biology is now well established, and its usefulness in combination with macromolecular crystallography is clear. However, the highly averaged SAXS data present a significant risk of over-interpretation to the unwary practitioner, and it can be challenging to frame SAXS results in a manner that maximises the reliability of the conclusions drawn. In this review, a series of recent examples are used to illustrate both the challenges for interpretation and approaches through which these can be overcome.

An optimized SEC-SAXS system enabling high X-ray dose for rapid SAXS assessment with correlated UV measurements for biomolecular structure analysis

A new optimized size exclusion chromatography small-angle X-ray scattering (SEC-SAXS) system for biomolecular SAXS at the Australian Synchrotron SAXS/WAXS beamline has been developed. The compact configuration reduces sample dilution to maximize sensitivity. Coflow sample presentation allows an 11-fold increase in flux on sample without capillary fouling, improving throughput and data quality, which are now primarily limited by the full flux available on the beamline. Multi-wavelength fibre optic UV analysis in close proximity to the X-ray beam allows for accurate concentration determination for samples with known UV extinction coefficients and thus estimation of the molecular weight of the scattering particle from the forward X-ray scattering intensity. Fast-flow low-volume SEC columns provide sample throughput competitive with batch concentration series measurements, albeit with a concomitant reduction of potential resolution relative to lower flow rates and larger SEC columns. The performance of the system is demonstrated using a set of model proteins, and its utility to solve various challenges is illustrated with a diverse suite of protein samples. These developments increase the quality and rigor of SEC-SAXS analysis and open new avenues for biomolecular solution SEC-SAXS studies that have been challenged by low sample yields, temporal instability, radiation sensitivity and complex mixtures.