Lorna C. Waters

Structure and Interactions of the Human Programmed Cell Death 1 Receptor

Background: The inhibitory leukocyte receptor PD-1 binds two ligands, PD-L1 and PD-L2. Results: Nuclear magnetic resonance analysis and rigorous binding and thermodynamic measurements reveal the structure of, and the mode of ligand recognition by, PD-1. Conclusion: PD-L1 and PD-L2 bind differently to PD-1 and much more weakly than expected. Significance: Potent inhibitory signaling can be initiated by weakly interacting receptors. PD-1, a receptor expressed by T cells, B cells, and monocytes, is a potent regulator of immune responses and a promising therapeutic target. The structure and interactions of human PD-1 are, however, incompletely characterized. We present the solution nuclear magnetic resonance (NMR)-based structure of the human PD-1 extracellular region and detailed analyses of its interactions with its ligands, PD-L1 and PD-L2. PD-1 has typical immunoglobulin superfamily topology but differs at the edge of the GFCC′ sheet, which is flexible and completely lacks a C″ strand. Changes in PD-1 backbone NMR signals induced by ligand binding suggest that, whereas binding is centered on the GFCC′ sheet, PD-1 is engaged by its two ligands differently and in ways incompletely explained by crystal structures of mouse PD-1·ligand complexes. The affinities of these interactions and that of PD-L1 with the costimulatory protein B7-1, measured using surface plasmon resonance, are significantly weaker than expected. The 3–4-fold greater affinity of PD-L2 versus PD-L1 for human PD-1 is principally due to the 3-fold smaller dissociation rate for PD-L2 binding. Isothermal titration calorimetry revealed that the PD-1/PD-L1 interaction is entropically driven, whereas PD-1/PD-L2 binding has a large enthalpic component. Mathematical simulations based on the biophysical data and quantitative expression data suggest an unexpectedly limited contribution of PD-L2 to PD-1 ligation during interactions of activated T cells with antigen-presenting cells. These findings provide a rigorous structural and biophysical framework for interpreting the important functions of PD-1 and reveal that potent inhibitory signaling can be initiated by weakly interacting receptors.

Structural diversity in p160/CREB-binding protein coactivator complexes.

Ligand-induced transcription by nuclear receptors involves the recruitment of p160 coactivators such as steroid receptor coactivator 1 (SRC1), in complex with histone acetyltransferases such as CREB-binding protein (CBP) and p300. Here we describe the solution structure of a complex formed by the SRC1 interaction domain (SID) of CBP and the activation domain (AD1) of SRC1, both of which contain four helical regions (Cα1, Cα2, Cα3, and Cα3′ in CBP and Sα1, Sα2′, Sα2, and Sα3 in SRC1). A tight four-helix bundle is formed between Sα1, Cα1, Cα2, and Cα3 that is capped by Sα3. In contrast to the structure of the AD1 domain of the related p160 protein ACTR in complex with CBP SID, the sequences forming Sα2′ and Sα2 in SRC1 AD1 are not involved in the interface between the two domains but rather serve to position Sα3. Thus, although the CBP SID domain adopts a similar fold in complex with different p160 proteins, the topologies of the AD1 domains are strikingly different, a feature that is likely to contribute to functional specificity of these coactivator complexes.

Solution structure of the Mycobacterium tuberculosis EsxG·EsxH complex: functional implications and comparisons with other M. tuberculosis Esx family complexes.

Mycobacterium tuberculosis encodes five type VII secretion systems that are responsible for exporting a number of proteins, including members of the Esx family, which have been linked to tuberculosis pathogenesis and survival within host cells. The gene cluster encoding ESX-3 is regulated by the availability of iron and zinc, and secreted protein products such as the EsxG·EsxH complex have been associated with metal ion acquisition. EsxG and EsxH have previously been shown to form a stable 1:1 heterodimeric complex, and here we report the solution structure of the complex, which features a core four-helix bundle decorated at both ends by long, highly flexible, N- and C-terminal arms that contain a number of highly conserved residues. Despite clear similarities in the overall backbone fold to the EsxA·EsxB complex, the structure reveals some striking differences in surface features, including a potential protein interaction site on the surface of the EsxG·EsxH complex. EsxG·EsxH was also found to contain a specific Zn2+ binding site formed from a cluster of histidine residues on EsxH, which are conserved across obligate mycobacterial pathogens including M. tuberculosis and Mycobacterium leprae. This site may reflect an essential role in zinc ion acquisition or point to Zn2+-dependent regulation of its interaction with functional partner proteins. Overall, the surface features of both the EsxG·EsxH and the EsxA·EsxB complexes suggest functions mediated via interactions with one or more target protein partners.

Structure of the C-terminal MA-3 domain of the tumour suppressor protein Pdcd4 and characterization of its interaction with eIF4A

Programmed cell death protein 4 (Pdcd4) is a novel tumour suppressor protein, which is involved in the control of eukaryotic transcription and translation. The regulation of translation involves specific interactions with eukaryotic initiation factor (eIF)4A and eIF4G, which are mediated via the two tandem MA-3 domains. We have determined the structure of the C-terminal MA-3 domain of Pdcd4 (Pdcd4 MA-3C), characterized its interaction with eIF4A and compared the features of nuclear magnetic resonance (NMR) spectra obtained from the single domain and tandem MA-3 region. Pdcd4 MA-3C is composed of three layers of helix–turn–helix hairpins capped by a single helix and shows close structural homology to the atypical HEAT repeats found in many eIFs. The sequence conservation and NMR data strongly suggest that the tandem MA-3 region is composed of two equivalent domains connected by a somewhat flexible linker. Pdcd4 MA-3C was found to interact with the N-terminal domain of eIF4A through a conserved surface region encompassing the loop connecting α5 and α6 and the turn linking α3 and α4. This site is strongly conserved in other MA-3 domains known to interact with eIF4A, including the preceding domain of Pdcd4, suggesting a common mode of binding.

Molecular features governing the stability and specificity of functional complex formation by Mycobacterium tuberculosis CFP-10/ESAT-6 family proteins.

The Mycobacterium tuberculosis complex CFP-10/ESAT-6 family proteins play essential but poorly defined roles in tuberculosis pathogenesis. In this article we report the results of detailed spectroscopic studies of several members of the CFP-10/ESAT-6 family. This work shows that the CFP-10/ESAT-6 related proteins, Rv0287 and Rv0288, form a tight 1:1 complex, which is predominantly helical in structure and is predicted to closely resemble the complex formed by CFP-10 and ESAT-6. In addition, the Rv0287·Rv0288 complex was found to be significantly more stable to both chemical and temperature induced denaturation than CFP-10·ESAT-6. This approach demonstrated that neither Rv0287·Rv0288 nor the CFP-10·ESAT-6 complexes are destabilized at low pH (4.5), indicating that even in low pH environments, such as the mature phagosome, both Rv0287·Rv0288 and CFP-10·ESAT-6 undoubtedly function as complexes rather than individual proteins. Analysis of the structure of the CFP-10·ESAT-6 complex and optimized amino acid sequence alignments of M. tuberculosis CFP-10/ESAT-6 family proteins revealed that residues involved in the intramolecular contacts between helices are conserved across the CFP-10/ESAT-6 family, but not those involved in primarily intermolecular contacts. This analysis identified the molecular basis for the specificity and stability of complex formation between CFP-10/ESAT-6 family proteins, and indicates that the formation of functional complexes with key roles in pathogenesis will be limited to genome partners, or very closely related family members, such as Rv0287/Rv0288 and Rv3019c/Rv3020c.

Mycobacterium tuberculosis RNA polymerase-binding protein A (RbpA) and its interactions with sigma factors.

Background: RNA polymerase-binding protein A (RbpA) plays an unknown essential role in Mycobacterium tuberculosis. Results: The structure of RbpA was solved using NMR. Conclusion: RbpA binds sigma factors A and B via its conserved N and C termini. Significance: The identified interactions shed light on the function of RbpA in regulating transcription. RNA polymerase-binding protein A (RbpA), encoded by Rv2050, is specific to the actinomycetes, where it is highly conserved. In the pathogen Mycobacterium tuberculosis, RbpA is essential for growth and survival. RbpA binds to the β subunit of the RNA polymerase where it activates transcription by unknown mechanisms, and it may also influence the response of M. tuberculosis to the current frontline anti-tuberculosis drug rifampicin. Here we report the solution structure of RbpA and identify the principle sigma factor σA and the stress-induced σB as interaction partners. The protein has a central ordered domain with a conserved hydrophobic surface that may be a potential protein interaction site. The N and C termini are highly dynamic and are involved in the interaction with the sigma factors. RbpA forms a tight complex with the N-terminal domain of σB via its N- and C-terminal regions. The interaction with sigma factors may explain how RbpA stabilizes sigma subunit binding to the core RNA polymerase and thereby promotes initiation complex formation. RbpA could therefore influence the competition between principal and alternative sigma factors and hence the transcription profile of the cell.

Tumor suppressor protein Pdcd4 interacts with Daxx and modulates the stability of Daxx and the Hipk2-dependent phosphorylation of p53 at serine 46.

The tumor suppressor protein Pdcd4 is a nuclear/cytoplasmic shuttling protein that has been implicated in the development of several types of human cancer. In the nucleus, Pdcd4 affects the transcription of specific genes by modulating the activity of several transcription factors. We have identified the Daxx protein as a novel interaction partner of Pdcd4. Daxx is a scaffold protein with roles in diverse processes, including transcriptional regulation, DNA-damage signaling, apoptosis and chromatin remodeling. We show that the interaction of both proteins is mediated by the N-terminal domain of Pdcd4 and the central part of Daxx, and that binding to Pdcd4 stimulates the degradation of Daxx, presumably by disrupting the interaction of Daxx with the de-ubiquitinylating enzyme Hausp. Daxx has previously been shown to serve as a scaffold for protein kinase Hipk2 and tumor suppressor protein p53 and to stimulate the phosphorylation of p53 at serine 46 (Ser-46) in response to genotoxic stress. We show that Pdcd4 also disrupts the Daxx–Hipk2 interaction and inhibits the phosphorylation of p53. We also show that ultraviolet irradiation decreases the expression of Pdcd4. Taken together, our results support a model in which Pdcd4 serves to suppress the phosphorylation of p53 in the absence of DNA damage, while the suppressive effect of Pdcd4 is abrogated after DNA damage owing to the decrease of Pdcd4. Overall, our data demonstrate that Pdcd4 is a novel modulator of Daxx function and provide evidence for a role of Pdcd4 in restraining p53 activity in unstressed cells.

Structure of the Tandem MA-3 Region of Pdcd4 Protein and Characterization of Its Interactions with eIF4A and eIF4G: MOLECULAR MECHANISMS OF A TUMOR SUPPRESSOR*

One of the key regulatory points of translation initiation is recruitment of the 43S preinitation complex to the 5′ mRNA cap by the eIF4F complex (eIF4A, eIF4E, and eIF4G). The tumor suppressor protein Pdcd4 has been shown to inhibit cap-dependent translation by interacting tightly with the RNA helicase eIF4A via its tandem MA-3 domains. The NMR studies reported here reveal a fairly extensive and well defined interface between the two MA-3 domains in solution, which appears to be stabilized by a network of interdomain salt bridges and hydrogen bonds, and reveals a unique orientation of the two domains. Characterization of the stoichiometry of the Pdcd4-eIF4A complex suggests that under physiological conditions Pdcd4 binds to a single molecule of eIF4A, which involves contacts with both Pdcd4 MA-3 domains. We also show that contacts mediated by a conserved acidic patch on the middle MA-3 domain of Pdcd4 are essential for forming a tight complex with eIF4A in vivo, whereas the equivalent region of the C-terminal MA-3 domain appears to have no role in complex formation in vivo. The formation of a 1:1 eIF4A-Pdcd4 complex in solution is consistent with the reported presence in vivo of only one molecule of eIF4A in the eIF4F complex. Pdcd4 has also been reported to interact directly with the middle region of eIF4G, however, we were unable to obtain any evidence for even a weak, transient direct interaction.

Conformational heterogeneity in antibody-protein antigen recognition: implications for high affinity protein complex formation.

Background: Antibodies are essential components of the immune system which recognize specific antigens with high affinity. Results: Protein antigen binding sites on antibodies show conformational exchange on a millisecond to second timescale. Conclusion: Conformational heterogeneity at high affinity protein-protein interaction sites may be common and facilitate efficient protein complex formation. Significance: High affinity protein-protein interactions are critical for many biological processes. Specific, high affinity protein-protein interactions lie at the heart of many essential biological processes, including the recognition of an apparently limitless range of foreign proteins by natural antibodies, which has been exploited to develop therapeutic antibodies. To mediate biological processes, high affinity protein complexes need to form on appropriate, relatively rapid timescales, which presents a challenge for the productive engagement of complexes with large and complex contact surfaces (∼600–1800 Å2). We have obtained comprehensive backbone NMR assignments for two distinct, high affinity antibody fragments (single chain variable and antigen-binding (Fab) fragments), which recognize the structurally diverse cytokines interleukin-1β (IL-1β, β-sheet) and interleukin-6 (IL-6, α-helical). NMR studies have revealed that the hearts of the antigen binding sites in both free anti-IL-1β Fab and anti-IL-6 single chain variable exist in multiple conformations, which interconvert on a timescale comparable with the rates of antibody-antigen complex formation. In addition, we have identified a conserved antigen binding-induced change in the orientation of the two variable domains. The observed conformational heterogeneity and slow dynamics at protein antigen binding sites appears to be a conserved feature of many high affinity protein-protein interfaces structurally characterized by NMR, suggesting an essential role in protein complex formation. We propose that this behavior may reflect a soft capture, protein-protein docking mechanism, facilitating formation of high affinity protein complexes on a timescale consistent with biological processes.

15 N, 13 C and 1 H resonance assignments and secondary structure determination of the Mycobacterium tuberculosis Rv0287-Rv0288 protein complex

Comprehensive sequence specific 1H, 15N, and 13C resonance assignments are reported for the Mycobacterium tuberculosis Rv0287–Rv0288 protein complex. Analysis of the chemical shift data obtained indicates that each protein in the complex contains two relatively long helical regions joined by an irregular loop.