PDZ domains are protein-protein interaction modules that recognize specific C-terminal sequences to assemble protein complexes in multicellular organisms. By scanning billions of random peptides, we ...accurately map binding specificity for approximately half of the over 330 PDZ domains in the human and Caenorhabditis elegans proteomes. The domains recognize features of the last seven ligand positions, and we find 16 distinct specificity classes conserved from worm to human, significantly extending the canonical two-class system based on position -2. Thus, most PDZ domains are not promiscuous, but rather are fine-tuned for specific interactions. Specificity profiling of 91 point mutants of a model PDZ domain reveals that the binding site is highly robust, as all mutants were able to recognize C-terminal peptides. However, many mutations altered specificity for ligand positions both close and far from the mutated position, suggesting that binding specificity can evolve rapidly under mutational pressure. Our specificity map enables the prediction and prioritization of natural protein interactions, which can be used to guide PDZ domain cell biology experiments. Using this approach, we predicted and validated several viral ligands for the PDZ domains of the SCRIB polarity protein. These findings indicate that many viruses produce PDZ ligands that disrupt host protein complexes for their own benefit, and that highly pathogenic strains target PDZ domains involved in cell polarity and growth.
The interface between antibody and antigen is often depicted as a lock and key, suggesting that an antibody surface can accommodate only one antigen. Here, we describe an antibody with an antigen ...binding site that binds two distinct proteins with high affinity. We isolated a variant of Herceptin, a therapeutic monoclonal antibody that binds the human epidermal growth factor receptor 2 (HER2), on the basis of its ability to simultaneously interact with vascular endothelial growth factor (VEGF). Crystallographic and mutagenesis studies revealed that distinct amino acids of this antibody, called bH1, engage HER2 and VEGF energetically, but there is extensive overlap between the antibody surface areas contacting the two antigens. An affinity-improved version of bH1 inhibits both HER2- and VEGF-mediated cell proliferation in vitro and tumor progression in mouse models. Such "two-in-one" antibodies challenge the monoclonal antibody paradigm of one binding site, one antigen. They could also provide new opportunities for antibody-based therapy.
PDZ (PSD-95/Discs-large/ZO1) domains are interaction modules that typically bind to specific C-terminal sequences of partner proteins and assemble signaling complexes in multicellular organisms. We ...have analyzed the existing database of PDZ domain structures in the context of a specificity tree based on binding specificities defined by peptide-phage binding selections. We have identified 16 structures of PDZ domains in complex with high-affinity ligands and have elucidated four additional structures to assemble a structural database that covers most of the branches of the PDZ specificity tree. A detailed comparison of the structures reveals features that are responsible for the diverse specificities across the PDZ domain family. Specificity differences can be explained by differences in PDZ residues that are in contact with the peptide ligands, but these contacts involve both side-chain and main-chain interactions. Most PDZ domains bind peptides in a canonical conformation in which the ligand main chain adopts an extended β-strand conformation by interacting in an antiparallel fashion with a PDZ β-strand. However, a subset of PDZ domains bind peptides with a bent main-chain conformation and the specificities of these non-canonical domains could not be explained based on canonical structures. Our analysis provides a structural portrait of the PDZ domain family, which serves as a guide in understanding the structural basis for the diverse specificities across the family.
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•We assembled a structural database that covers the major PDZ domain specificities.•The structural database reveals features responsible for diverse PDZ specificities.•PDZ domain specificity depends on both main-chain and side-chain interactions.
While the p90 ribosomal S6 kinase (RSK) family has been implicated in multiple tumor cell functions, the full understanding of this kinase family has been restricted by the lack of highly selective ...inhibitors. A bis-phenol pyrazole was identified from high-throughput screening as an inhibitor of the N-terminal kinase of RSK2. Structure-based drug design using crystallography, conformational analysis, and scaffold morphing resulted in highly optimized difluorophenol pyridine inhibitors of the RSK kinase family as demonstrated cellularly by the inhibition of YB1 phosphorylation. These compounds provide for the first time in vitro tools with an improved selectivity and potency profile to examine the importance of RSK signaling in cancer cells and to fully evaluate RSK as a therapeutic target.
To examine the association between ultrasound (US)-detected knee inflammation and intermittent and constant pain experiences in patients with knee osteoarthritis (OA).
Participants with ...radiographically early-stage (Kellgren-Lawrence arthritis grading scale KL ≤ 2) and late-stage (KL ≥ 3) disease and frequent symptoms underwent musculoskeletal US measures of inflammation using the Outcome Measures in Rheumatology (OMERACT) knee US scoring system. Pain experiences were captured using the Measure of Intermittent and Constant Osteoarthritis Pain (ICOAP) tool. We assessed the association between US-synovitis and ICOAP pain experiences using a series of linear, logistic, or multinomial logistic regression models (as appropriate for each variable), while adjusting for age, sex, BMI, and radiographic stage. Secondary analyses were performed similarly by radiographic stage.
Pain and synovitis measures from 248 patients (453 knees) were included. Worse synovitis was associated with higher ICOAP constant pain scores (β 8.05, 95% CI 0.67-15.43), but not intermittent pain scores. Moderate-to-severe synovitis was associated with a 4.73-fold increased relative risk (95% CI 1.06-17.00) of a constant pain pattern. In secondary analyses, moderate-to-severe synovitis in early radiographic OA was associated with 2.70-higher odds (95% CI 1.04-7.02) of any constant pain, 3.28-higher odds (95% CI 1.43-7.52) of any intermittent pain, and with higher intermittent (β 10.47, 95% CI 1.03-19.91) and constant (β 12.62, 95% CI 3.02-22.23) pain scores. No associations were identified for synovitis in those with late radiographic OA.
In patients with knee OA, moderate-to-severe synovitis is most strongly associated with constant pain. Inflammation may play context-specific roles across pain experiences, especially in earlier radiographic stages of knee OA.
To identify the presence and distribution of histopathological features of synovial inflammation and tissue damage, and to test their associations with ultrasound (US) imaging measures of synovitis ...and patient-reported measures of pain in knee osteoarthritis (OA).
In the cross-sectional study of 122 patients undergoing surgery for painful late-stage (Kellgren-Lawrence Grade 3 or 4) knee OA, we compared US measures of synovitis (n = 118) and pain (Knee Injury and Osteoarthritis Outcome Score) to histopathological measures of inflammation vs. synovial tissue damage in synovial tissue biopsies. Associations of histopathological features with US measures of inflammation or pain were assessed using linear or logistic regression while controlling for covariates.
Histopathological features of inflammation were associated with higher odds of moderate/severe US synovitis (odds ratio OR = 1.34 95%CI 1.04, 1.74), whereas features of synovial tissue damage were associated with lower odds of moderate/severe US synovitis (OR = 0.77 95%CI 0.57, 1.03). Worse histopathological scores for synovial tissue damage were associated with more pain (−1.47 95%CI −2.88, −0.05), even while adjusting for synovial inflammation (−1.61 95%CI −3.12, −0.10).
Synovial tissue damage is associated with pain in late-stage knee OA, independent from inflammation and radiographic damage. These novel findings suggest that preventing synovial tissue damage may be an important goal of disease-modifying OA therapy.
Abstract
The brightest observed emission line in many star-forming galaxies is the
158
μ
m line, making it detectable up to
z
∼ 7. In order to better understand and quantify the
emission as a ...tracer of star formation, the theoretical ratio between the
205
μ
m emission and the
158
μ
m emission has been employed to empirically determine the fraction of
emission that originates from the ionized and neutral phases of the interstellar medium (ISM). Sub-kiloparsec measurements of the
158
μ
m and
205
μ
m lines in nearby galaxies have recently become available as part of the Key Insights in Nearby Galaxies: a Far Infrared Survey with
Herschel
(KINGFISH) and Beyond the Peak programs. With the information from these two far-infrared lines along with the multi-wavelength suite of KINGFISH data, a calibration of the
emission line as a star formation rate (SFR) indicator and a better understanding of the
deficit are pursued.
emission is also compared to polycyclic aromatic hydrocarbon (PAH) emission in these regions to compare photoelectric heating from PAH molecules to cooling by
in the neutral and ionized phases of the ISM. We find that the
emission originating in the neutral phase of the ISM does not exhibit a deficit with respect to the infrared luminosity and is therefore preferred over the
emission originating in the ionized phase of the ISM as an SFR indicator for the normal star-forming galaxies included in this sample.
Members of the interleukin-1 (IL-1) family of cytokines play major roles in host defense and immune system regulation in infectious and inflammatory diseases. IL-1 cytokines trigger a biological ...response in effector cells by assembling a heterotrimeric signaling complex with two IL-1 receptor chains, a high-affinity primary receptor and a low-affinity coreceptor. To gain insights into the signaling mechanism of the novel IL-1-like cytokine IL-33, we first solved its solution structure and then performed a detailed biochemical and structural characterization of the interaction between IL-33, its primary receptor ST2, and the coreceptor IL-1RAcP. Using nuclear magnetic resonance data, we obtained a model of the IL-33/ST2 complex in solution that is validated by small-angle X-ray scattering (SAXS) data and is similar to the IL-1β/IL-1R1 complex. We extended our SAXS analysis to the IL-33/ST2/IL-1RAcP and IL-1β/IL-1R1/IL-1RAcP complexes and propose a general model of the molecular architecture of IL-1 ternary signaling complexes.
Dishevelled proteins are key regulators of Wnt signaling pathways that have been implicated in the progression of human cancers. We found that the binding cleft of the Dishevelled PDZ domain is more ...flexible than those of canonical PDZ domains and enables recognition of both C-terminal and internal peptides. These peptide ligands inhibit Wnt/β-catenin signaling in cells, showing that Dishevelled PDZ domains are potential targets for small-molecule cancer therapeutics.
We report a comprehensive analysis of sequence features that allow for the production of autonomous human heavy chain variable (VH) domains that are stable and soluble in the absence of a light chain ...partner. Using combinatorial phage-displayed libraries and conventional biophysical methods, we analyzed the entire former light chain interface and the third complementarity determining region (CDR3). Unlike the monomeric variable domains of camelid heavy chain antibodies (VHH domains), in which autonomous behavior depends on interactions between the hydrophobic former light chain interface and CDR3, we find that the stability of many in vitro evolved VH domains is essentially independent of the CDR3 sequence and instead derives from mutations that increase the hydrophilicity of the former light chain interface by replacing exposed hydrophobic residues with structurally compatible hydrophilic substitutions. The engineered domains can be expressed recombinantly at high yield, are predominantly monomeric at high concentrations, unfold reversibly, and are even more thermostable than typical camelid VHH domains. Many of the stabilizing mutations are rare in natural VH and VHH domains and thus could not be predicted by studying natural sequences and structures. The results demonstrate that autonomous VH domains with structural properties beyond the scope of natural frameworks can be derived by using non-natural mutations, which differ from those found in camelid VHH domains. These findings should enable the development of libraries of synthetic VH domains with CDR3 diversities unconstrained by structural demands.
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![Using [C ii] 158 μm Emissio...](http://api.summon.serialssolutions.com/2.0.0/image/issn/XR4PS5YY4K/0004-637X_1538-4357/small "Using [C ii] 158 μm Emission from Isolated ISM Phases as a Star Formation Rate Indicator")
