Jiansheng Jiang

RAGE is a nucleic acid receptor that promotes inflammatory responses to DNA

Receptor for advanced glycation end-products (RAGE) detects nucleic acids and promotes DNA uptake into endosomes, which in turn lowers the immune recognition threshold for TLR9 activation.

Structure of the Absent in Melanoma 2 (AIM2) Pyrin Domain Provides Insights into the Mechanisms of AIM2 Autoinhibition and Inflammasome Assembly

Background: AIM2 binds dsDNA and associates with ASC through their PYDs to form an inflammasome. Results: The AIM2 PYD structure illustrates distinct charge distribution and a unique hydrophobic patch. Conclusion: The AIM2 PYD may bind the ASC PYD and the AIM2 HIN domain through overlapping surface. Significance: These findings provide insights into the mechanisms of AIM2 autoinhibition and inflammasome assembly. Absent in melanoma 2 (AIM2) is a cytosolic double-stranded (dsDNA) sensor essential for innate immune responses against DNA viruses and bacteria such as Francisella and Listeria. Upon dsDNA engagement, the AIM2 amino-terminal pyrin domain (PYD) is responsible for downstream signaling to the adapter protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) through homotypic PYD-PYD interactions and the assembly of an inflammasome. Toward a better understanding of the AIM2 signaling mechanism, we determined the crystal structure of the human AIM2 PYD. The structure reveals a death domain fold with a short α3 helix that is buttressed by a highly conserved lysine residue at the α2 helix, which may stabilize the α3 helix for potential interaction with partner domains. The surface of the AIM2 PYD exhibits distinct charge distribution with highly acidic α1-α2 helices and highly basic α5-α6 helices. A prominent solvent-exposed hydrophobic patch formed by residues Phe-27 and Phe-28 at the α2 helix resembles a similar surface involved in the death effector domain homotypic interactions. Docking studies suggest that the AIM2 PYD may bind the AIM2 hematopoietic interferon-inducible nuclear (HIN) domain or ASC PYD using overlapping surface near the α2 helix. This may ensure that AIM2 interacts with the downstream adapter ASC only upon release of the autoinhibition by the dsDNA ligand. Our work thus unveils novel structural features of the AIM2 PYD and provides insights into the potential mechanisms of the PYD-HIN and PYD-PYD interactions important for AIM2 autoinhibition and inflammasome assembly.

Molecular mechanisms for the subversion of MyD88 signaling by TcpC from virulent uropathogenic Escherichia coli.

The Toll/IL-1 receptor (TIR) domains are crucial signaling modules during innate immune responses involving the Toll-like receptors (TLRs) and IL-1 receptor (IL-1R). Myeloid differential factor 88 (MyD88) is a central TIR domain-containing adapter molecule responsible for nearly all TLR-mediated signaling and is targeted by a TIR domain-containing protein C (TcpC) from virulent uropathogenic Escherichia coli, a common human pathogen. The mechanism of such molecular antagonism has remained elusive. We present the crystal structure of the MyD88 TIR domain with distinct loop conformations that underscore the functional specialization of the adapter, receptor, and microbial TIR domains. Our structural analyses shed light on the genetic mutations at these loops as well as the Poc site. We demonstrate that TcpC directly associates with MyD88 and TLR4 through its predicted DD and BB loops to impair the TLR-induced cytokine induction. Furthermore, NMR titration experiments identify the unique CD, DE, and EE loops from MyD88 at the TcpC-interacting surface, suggesting that TcpC specifically engages these MyD88 structural elements for immune suppression. These findings thus provide a molecular basis for the subversion of TLR signaling by the uropathogenic E. coli virulence factor TcpC and furnish a framework for the design of novel therapeutic agents that modulate immune activation.

Crystal structures of the Toll/Interleukin-1 receptor (TIR) domains from the Brucella protein TcpB and host adaptor TIRAP reveal mechanisms of molecular mimicry.

Background: The Toll/IL-1 receptor (TIR) domains are crucial innate immune signaling modules. Results: The crystal structures of the TIR domains from TcpB and TIRAP reveal similar folds and distinct features. Conclusion: TcpB may mimic the function of TIRAP through their similar TIR domain structures. Significance: These findings suggest mechanisms of bacterial mimicry of host signaling adaptor proteins. The Toll/IL-1 receptor (TIR) domains are crucial innate immune signaling modules. Microbial TIR domain-containing proteins inhibit Toll-like receptor (TLR) signaling through molecular mimicry. The TIR domain-containing protein TcpB from Brucella inhibits TLR signaling through interaction with host adaptor proteins TIRAP/Mal and MyD88. To characterize the microbial mimicry of host proteins, we have determined the X-ray crystal structures of the TIR domains from the Brucella protein TcpB and the host adaptor protein TIRAP. We have further characterized homotypic interactions of TcpB using hydrogen/deuterium exchange mass spectrometry and heterotypic TcpB and TIRAP interaction by co-immunoprecipitation and NF-κB reporter assays. The crystal structure of the TcpB TIR domain reveals the microtubule-binding site encompassing the BB loop as well as a symmetrical dimer mediated by the DD and EE loops. This dimerization interface is validated by peptide mapping through hydrogen/deuterium exchange mass spectrometry. The human TIRAP TIR domain crystal structure reveals a unique N-terminal TIR domain fold containing a disulfide bond formed by Cys89 and Cys134. A comparison between the TcpB and TIRAP crystal structures reveals substantial conformational differences in the region that encompasses the BB loop. These findings underscore the similarities and differences in the molecular features found in the microbial and host TIR domains, which suggests mechanisms of bacterial mimicry of host signaling adaptor proteins, such as TIRAP.

Structure of the NLRP1 caspase recruitment domain suggests potential mechanisms for its association with procaspase‐1

The NLRP1 inflammasome responds to microbial challenges such as Bacillus anthracis infection and is implicated in autoimmune disease such as vitiligo. Human NLRP1 contains both an N‐terminal pyrin domain (PYD) and a C‐terminal caspase recruitment domain (CARD), with the latter being essential for its association with the downstream effector procaspase‐1. Here we report a 2.0 Å crystal structure of the human NLRP1 CARD as a fusion with the maltose‐binding protein. The structure reveals the six‐helix bundle fold of the NLRP1 CARD, typical of the death domain superfamily. The charge surface of the NLRP1 CARD structure and a procaspase‐1 CARD model suggests potential mechanisms for their association through electrostatic attraction. Proteins 2013; 81:1266–1270.

The Catalytic Subunit of the SWR1 Remodeler Is a Histone Chaperone for the H2A.Z-H2B Dimer

Histone variant H2A.Z-containing nucleosomes exist at most eukaryotic promoters and play important roles in gene transcription and genome stability. The multisubunit nucleosome-remodeling enzyme complex SWR1, conserved from yeast to mammals, catalyzes the ATP-dependent replacement of histone H2A in canonical nucleosomes with H2A.Z. How SWR1 catalyzes the replacement reaction is largely unknown. Here, we determined the crystal structure of the N-terminal region (599-627) of the catalytic subunit Swr1, termed Swr1-Z domain, in complex with the H2A.Z-H2B dimer at 1.78 Å resolution. The Swr1-Z domain forms a 310 helix and an irregular chain. A conserved LxxLF motif in the Swr1-Z 310 helix specifically recognizes the αC helix of H2A.Z. Our results show that the Swr1-Z domain can deliver the H2A.Z-H2B dimer to the DNA-(H3-H4)2 tetrasome to form the nucleosome by a histone chaperone mechanism.

Crystal structure of a TAPBPR-MHC I complex reveals the mechanism of peptide editing in antigen presentation.

Two snapshots of the TAPBPR-MHC I complex Cytotoxic CD8+ T cells recognize infected and cancerous cells by scrutinizing the antigenic peptides presented by the major histocompatibility complex class I (MHC I). Peptide binding and exchange occurs in the endoplasmic reticulum in a sequence of events mediated by the chaperones tapasin and TAPBPR (see the Perspective by Cresswell). Thomas and Tampé resolved the crystal structure of the TAPBPR-MHC I editing complex by using a photocleavable high-affinity peptide to stabilize the MHC molecule. Jiang et al. crystalized MHC I molecules inhabited by truncated disulfide-linked peptides that still permit TAPBPR to bind. These complimentary snapshots elucidate the dynamic process by which chaperones stabilize the groove of peptide-free MHC I molecules. This helps MHC I sample peptide candidates and facilitates the generation of peptide repertoires enriched with high-affinity antigenic peptides. Science, this issue p. 1060, p. 1064; see also p. 992 Two different approaches yield complimentary structures of TAPBR in complex with MHC I. Central to CD8+ T cell–mediated immunity is the recognition of peptide–major histocompatibility complex class I (p–MHC I) proteins displayed by antigen-presenting cells. Chaperone-mediated loading of high-affinity peptides onto MHC I is a key step in the MHC I antigen presentation pathway. However, the structure of MHC I with a chaperone that facilitates peptide loading has not been determined. We report the crystal structure of MHC I in complex with the peptide editor TAPBPR (TAP-binding protein–related), a tapasin homolog. TAPBPR remodels the peptide-binding groove of MHC I, resulting in the release of low-affinity peptide. Changes include groove relaxation, modifications of key binding pockets, and domain adjustments. This structure captures a peptide-receptive state of MHC I and provides insights into the mechanism of peptide editing by TAPBPR and, by analogy, tapasin.

Interaction of TAPBPR, a tapasin homolog, with MHC-I molecules promotes peptide editing

Significance This report explores the biochemical and structural basis of the interactions of TAP binding protein, related (TAPBPR), a tapasin homolog, with MHC-I molecules. TAPBPR associates with MHC-I molecules early in their biosynthesis and folding but is not part of the peptide-loading complex (PLC). Here, by examining the interactions of recombinant TAPBPR with peptide-free and peptide-complexed MHC-I molecules, we show that TAPBPR serves as a peptide editor. Structural comparison of TAPBPR with tapasin indicates the similarities of the two molecules and provides a basis for evaluating the steps of peptide loading. Understanding the molecular underpinnings of peptide loading of MHC-I by TAPBPR and tapasin has wide-ranging influence on our ability to modulate peptide loading for vaccine design and T-cell recognition. Peptide loading of major histocompatibility complex class I (MHC-I) molecules is central to antigen presentation, self-tolerance, and CD8+ T-cell activation. TAP binding protein, related (TAPBPR), a widely expressed tapasin homolog, is not part of the classical MHC-I peptide-loading complex (PLC). Using recombinant MHC-I molecules, we show that TAPBPR binds HLA-A*02:01 and several other MHC-I molecules that are either peptide-free or loaded with low-affinity peptides. Fluorescence polarization experiments establish that TAPBPR augments peptide binding by MHC-I. The TAPBPR/MHC-I interaction is reversed by specific peptides, related to their affinity. Mutational and small-angle X-ray scattering (SAXS) studies confirm the structural similarities of TAPBPR with tapasin. These results support a role of TAPBPR in stabilizing peptide-receptive conformation(s) of MHC-I, permitting peptide editing.

An allosteric site in the T-cell receptor C beta domain plays a critical signalling role.

The molecular mechanism through which the interaction of a clonotypic αβ T-cell receptor (TCR) with a peptide-loaded major histocompatibility complex (p/MHC) leads to T-cell activation is not yet fully understood. Here we exploit a high-affinity TCR (B4.2.3) to examine the structural changes that accompany binding to its p/MHC ligand (P18-I10/H2-Dd). In addition to conformational changes in complementarity-determining regions (CDRs) of the TCR seen in comparison of unliganded and bound X-ray structures, NMR characterization of the TCR β-chain dynamics reveals significant chemical shift effects in sites removed from the MHC-binding site. Remodelling of electrostatic interactions near the Cβ H3 helix at the membrane-proximal face of the TCR, a region implicated in interactions with the CD3 co-receptor, suggests a possible role for an allosteric mechanism in TCR signalling. The contribution of these TCR residues to signal transduction is supported by mutagenesis and T-cell functional assays.

Design of an expression system to enhance MBP-mediated crystallization

Crystallization chaperones have been used to facilitate the crystallization of challenging proteins. Even though the maltose-binding protein (MBP) is one of the most commonly used crystallization chaperones, the design of optimal expression constructs for crystallization of MBP fusion proteins remains a challenge. To increase the success rate of MBP-facilitated crystallization, a series of expression vectors have been designed with either a short flexible linker or a set of rigid helical linkers. Seven death domain superfamily members were tested for crystallization with this set of vectors, six of which had never been crystallized before. All of the seven targets were crystallized, and their structures were determined using at least one of the vectors. Our successful crystallization of all of the targets demonstrates the validity of our approach and expands the arsenal of the crystallization chaperone toolkit, which may be applicable to crystallization of other difficult protein targets, as well as to other crystallization chaperones.