Antibody discovery identifies regulatory mechanisms of protein arginine deiminase 4.

Unlocking the potential of protein arginine deiminase 4 (PAD4) as a drug target for rheumatoid arthritis requires a deeper understanding of its regulation. In this study, we use unbiased antibody selections to identify functional antibodies capable of either activating or inhibiting PAD4 activity. Through cryogenic-electron microscopy, we characterized the structures of these antibodies in complex with PAD4 and revealed insights into their mechanisms of action. Rather than steric occlusion of the substrate-binding catalytic pocket, the antibodies modulate PAD4 activity through interactions with allosteric binding sites adjacent to the catalytic pocket. These binding events lead to either alteration of the active site conformation or the enzyme oligomeric state, resulting in modulation of PAD4 activity. Our study uses antibody engineering to reveal new mechanisms for enzyme regulation and highlights the potential of using PAD4 agonist and antagonist antibodies for studying PAD4-dependency in disease models and future therapeutic development.

Computational pipeline provides mechanistic understanding of Omicron variant of concern neutralizing engineered ACE2 receptor traps.

The SARS-CoV-2 Omicron variant, with 15 mutations in Spike receptor-binding domain (Spike-RBD), renders virtually all clinical monoclonal antibodies against WT SARS-CoV-2 ineffective. We recently engineered the SARS-CoV-2 host entry receptor, ACE2, to tightly bind WT-RBD and prevent viral entry into host cells ("receptor traps"). Here we determine cryo-EM structures of our receptor traps in complex with stabilized Spike ectodomain. We develop a multi-model pipeline combining Rosetta protein modeling software and cryo-EM to allow interface energy calculations even at limited resolution and identify interface side chains that allow for high-affinity interactions between our ACE2 receptor traps and Spike-RBD. Our structural analysis provides a mechanistic rationale for the high-affinity (0.53-4.2 nM) binding of our ACE2 receptor traps to Omicron-RBD confirmed with biolayer interferometry measurements. Finally, we show that ACE2 receptor traps potently neutralize Omicron and Delta pseudotyped viruses, providing alternative therapeutic routes to combat this evolving virus.

Computational pipeline provides mechanistic understanding of Omicron variant of concern neutralizing engineered ACE2 receptor traps.

The SARS-CoV-2 Omicron variant, with 15 mutations in Spike receptor binding domain (Spike-RBD), renders virtually all clinical monoclonal antibodies against WT SARS-CoV-2 ineffective. We recently engineered the SARS-CoV-2 host entry receptor, ACE2, to tightly bind WT-Spike-RBD and prevent viral entry into host cells ("receptor traps"). Here we determine cryo-EM structures of our receptor traps in complex with full length Spike. We develop a multi-model pipeline combining Rosetta protein modeling software and cryo-EM to allow interface energy calculations even at limited resolution and identify interface side chains that allow for high affinity interactions between our ACE2 receptor traps and Spike-RBD. Our structural analysis provides a mechanistic rationale for the high affinity (0.53 - 4.2nM) binding of our ACE2 receptor traps to Omicron-RBD confirmed with biolayer interferometry measurements. Finally, we show that ACE2 receptor traps potently neutralize Omicron- and Delta-pseudotyped viruses, providing alternative therapeutic routes to combat this evolving virus.

Targeting a proteolytic neoepitope on CUB domain containing protein 1 (CDCP1) for RAS-driven cancers.

Extracellular proteolysis is frequently dysregulated in disease and can generate proteoforms with unique neoepitopes not found in healthy tissue. Here, we demonstrate that Abs that selectively recognize a proteolytic neoepitope on CUB domain containing protein 1 (CDCP1) could enable more effective and safer treatments for solid tumors. CDCP1 is highly overexpressed in RAS-driven cancers, and its ectodomain is cleaved by extracellular proteases. Biochemical, biophysical, and structural characterization revealed that the 2 cleaved fragments of CDCP1 remain tightly associated with minimal proteolysis-induced conformational change. Using differential phage display, we generated recombinant Abs that are exquisitely selective to cleaved CDCP1 with no detectable binding to the uncleaved form. These Abs potently targeted cleaved CDCP1-expressing cancer cells as an Ab-drug conjugate, an Ab-radionuclide conjugate, and a bispecific T cell engager. In a syngeneic pancreatic tumor model, these cleaved-specific Abs showed tumor-specific localization and antitumor activity with superior safety profiles compared with a pan-CDCP1 approach. Targeting proteolytic neoepitopes could provide an orthogonal "AND" gate for improving the therapeutic index.

Nucleoid remodeling during environmental adaptation is regulated by HU-dependent DNA bundling.

Bacterial nucleoid remodeling dependent on conserved histone-like protein, HU is one of the determining factors in global gene regulation. By imaging of near-native, unlabeled E. coli cells by soft X-ray tomography, we show that HU remodels nucleoids by promoting the formation of a dense condensed core surrounded by less condensed isolated domains. Nucleoid remodeling during cell growth and environmental adaptation correlate with pH and ionic strength controlled molecular switch that regulated HUαα dependent intermolecular DNA bundling. Through crystallographic and solution-based studies we show that these effects mechanistically rely on HUαα promiscuity in forming multiple electrostatically driven multimerization interfaces. Changes in DNA bundling consequently affects gene expression globally, likely by constrained DNA supercoiling. Taken together our findings unveil a critical function of HU-DNA interaction in nucleoid remodeling that may serve as a general microbial mechanism for transcriptional regulation to synchronize genetic responses during the cell cycle and adapt to changing environments.

Switchable resolution in soft x-ray tomography of single cells.

The diversity of living cells, in both size and internal complexity, calls for imaging methods with adaptable spatial resolution. Soft x-ray tomography (SXT) is a three-dimensional imaging technique ideally suited to visualizing and quantifying the internal organization of single cells of varying sizes in a near-native state. The achievable resolution of the soft x-ray microscope is largely determined by the objective lens, but switching between objectives is extremely time-consuming and typically undertaken only during microscope maintenance procedures. Since the resolution of the optic is inversely proportional to the depth of focus, an optic capable of imaging the thickest cells is routinely selected. This unnecessarily limits the achievable resolution in smaller cells and eliminates the ability to obtain high-resolution images of regions of interest in larger cells. Here, we describe developments to overcome this shortfall and allow selection of microscope optics best suited to the specimen characteristics and data requirements. We demonstrate that switchable objective capability advances the flexibility of SXT to enable imaging cells ranging in size from bacteria to yeast and mammalian cells without physically modifying the microscope, and we demonstrate the use of this technology to image the same specimen with both optics.

A molecular switch in mouse CD1d modulates natural killer T cell activation by α-galactosylsphingamides.

Type I natural killer T (NKT) cells are a population of innate like T lymphocytes that rapidly respond to α-GalCer presented by CD1d via the production of both pro- and anti-inflammatory cytokines. While developing novel α-GalCer analogs that were meant to be utilized as potential adjuvants because of their production of pro-inflammatory cytokines (Th1 skewers), we generated α-galactosylsphingamides (αGSA). Surprisingly, αGSAs are not potent antigens in vivo despite their strong T-cell receptor (TCR)-binding affinities. Here, using surface plasmon resonance (SPR), antigen presentation assays, and X-ray crystallography (yielding crystal structures of 19 different binary (CD1d-glycolipid) or ternary (CD1d-glycolipid-TCR) complexes at resolutions between 1.67 and 2.85 Å), we characterized the biochemical and structural details of αGSA recognition by murine NKT cells. We identified a molecular switch within murine (m)CD1d that modulates NKT cell activation by αGSAs. We found that the molecular switch involves a hydrogen bond interaction between Tyr-73 of mCD1d and the amide group oxygen of αGSAs. We further established that the length of the acyl chain controls the positioning of the amide group with respect to the molecular switch and works synergistically with Tyr-73 to control NKT cell activity. In conclusion, our findings reveal important mechanistic insights into the presentation and recognition of glycolipids with polar moieties in an otherwise apolar milieu. These observations may inform the development αGSAs as specific NKT cell antagonists to modulate immune responses.

Functional Relevance of Interleukin-1 Receptor Inter-domain Flexibility for Cytokine Binding and Signaling.

The interleukin 1 (IL-1) receptor family, whose members contain three immunoglobulin-like domains (D1-D3) in the extracellular region, is responsible for transmitting pleiotropic signals of IL-1 cytokines. The inter-domain flexibility of IL-1 receptors and its functional roles have not been fully elucidated. In this study, we used small-angle X-ray scattering to show that ligand-binding primary receptors and co-receptors in the family all have inherent inter-domain flexibility due to the D2/D3 linker. Variants of the IL-1RAcP and IL-18Rß co-receptors with mutated D2/D3 linkers cannot form a cytokine-receptor complex and mediate signaling. Our analysis further revealed that these mutated co-receptors exhibited a changed conformational ensemble, suggesting that loss of function is due to the alteration of receptor dynamics. Taken together, our results demonstrate that the D2/D3 linker is a critical functional determinant of IL-1 receptor and underscore the important roles of the inter-domain flexibility in cytokine/receptor binding and signaling.

X-ray radiolytic labeling reveals the molecular basis of orange carotenoid protein photoprotection and its interactions with fluorescence recovery protein.

In cyanobacterial photoprotection, the orange carotenoid protein (OCP) is photoactivated under excess light conditions and binds to the light-harvesting antenna, triggering the dissipation of captured light energy. In low light, the OCP relaxes to the native state, a process that is accelerated in the presence of fluorescence recovery protein (FRP). Despite the importance of the OCP in photoprotection, the precise mechanism of photoactivation by this protein is not well-understood. Using time-resolved X-ray-mediated in situ hydroxyl radical labeling, we probed real-time solvent accessibility (SA) changes at key OCP residues during photoactivation and relaxation. We observed a biphasic photoactivation process in which carotenoid migration preceded domain dissociation. We also observed a multiphasic relaxation process, with collapsed domain association preceding the final conformational rearrangement of the carotenoid. Using steady-state hydroxyl radical labeling, we identified sites of interaction between the FRP and OCP. In combination, the findings in this study provide molecular-level insights into the factors driving structural changes during OCP-mediated photoprotection in cyanobacteria, and furnish a basis for understanding the physiological relevance of the FRP-mediated relaxation process.

Conformational Plasticity of the Immunoglobulin Fc Domain in Solution.

Fragment crystallizable (Fc) region of immunoglobulin G (IgG) antibody binds to specific Fc receptors (FcγRs) to control antibody effector functions. Currently, engineered specific Fc-FcγR interactions are validated with a static conformation derived from the crystal structure. However, computational evidence suggests that the conformational variability of Fcs plays an important role in receptor recognition. Here we elucidate Fc flexibility of IgG1, IgG2, and IgG1 Fc with mutations (M255Y/S257T/T259E) in solution by small-angle X-ray scattering (SAXS). Measured SAXS profiles and experimental parameters show variations in flexibility between Fc isotypes. We develop and apply a modeling tool for an accurate conformational sampling of Fcs followed by SAXS fitting. Revealed conformational variability of the CH2 domain as low as 10 Å in displacement, illustrates the power of the atomistic modeling combined with SAXS. This inexpensive SAXS-based approach offers to improve the engineering of antibodies for tailoring Fc receptor interactions through altering and measuring Fc flexibility.

Galactosylsphingamides: new α-GalCer analogues to probe the F'-pocket of CD1d.

Invariant Natural Killer T-cells (iNKT-cells) are an attractive target for immune response modulation, as upon CD1d-mediated stimulation with KRN7000, a synthetic α-galactosylceramide, they produce a vast amount of cytokines. Here we present a synthesis that allows swift modification of the phytosphingosine side chain by amidation of an advanced methyl ester precursor. The resulting KRN7000 derivatives, termed α-galactosylsphingamides, were evaluated for their capacity to stimulate iNKT-cells. While introduction of the amide-motif in the phytosphingosine chain is tolerated for CD1d binding and TCR recognition, the studied α-galactosylsphingamides showed compromised antigenic properties.

Unconventional Peptide Presentation by Major Histocompatibility Complex (MHC) Class I Allele HLA-A*02:01: BREAKING CONFINEMENT.

Peptide antigen presentation by major histocompatibility complex (MHC) class I proteins initiates CD8+ T cell-mediated immunity against pathogens and cancers. MHC I molecules typically bind peptides with 9 amino acids in length with both ends tucked inside the major A and F binding pockets. It has been known for a while that longer peptides can also bind by either bulging out of the groove in the middle of the peptide or by binding in a zigzag fashion inside the groove. In a recent study, we identified an alternative binding conformation of naturally occurring peptides from Toxoplasma gondii bound by HLA-A*02:01. These peptides were extended at the C terminus (PΩ) and contained charged amino acids not more than 3 residues after the anchor amino acid at PΩ, which enabled them to open the F pocket and expose their C-terminal extension into the solvent. Here, we show that the mechanism of F pocket opening is dictated by the charge of the first charged amino acid found within the extension. Although positively charged amino acids result in the Tyr-84 swing, amino acids that are negatively charged induce a not previously described Lys-146 lift. Furthermore, we demonstrate that the peptides with alternative binding modes have properties that fit very poorly to the conventional MHC class I pathway and suggest they are presented via alternative means, potentially including cross-presentation via the MHC class II pathway.

An Intrinsically Disordered APLF Links Ku, DNA-PKcs, and XRCC4-DNA Ligase IV in an Extended Flexible Non-homologous End Joining Complex.

DNA double-strand break (DSB) repair by non-homologous end joining (NHEJ) in human cells is initiated by Ku heterodimer binding to a DSB, followed by recruitment of core NHEJ factors including DNA-dependent protein kinase catalytic subunit (DNA-PKcs), XRCC4-like factor (XLF), and XRCC4 (X4)-DNA ligase IV (L4). Ku also interacts with accessory factors such as aprataxin and polynucleotide kinase/phosphatase-like factor (APLF). Yet, how these factors interact to tether, process, and ligate DSB ends while allowing regulation and chromatin interactions remains enigmatic. Here, small angle X-ray scattering (SAXS) and mutational analyses show APLF is largely an intrinsically disordered protein that binds Ku, Ku/DNA-PKcs (DNA-PK), and X4L4 within an extended flexible NHEJ core complex. X4L4 assembles with Ku heterodimers linked to DNA-PKcs via flexible Ku80 C-terminal regions (Ku80CTR) in a complex stabilized through APLF interactions with Ku, DNA-PK, and X4L4. Collective results unveil the solution architecture of the six-protein complex and suggest cooperative assembly of an extended flexible NHEJ core complex that supports APLF accessibility while possibly providing flexible attachment of the core complex to chromatin. The resulting dynamic tethering furthermore, provides geometric access of L4 catalytic domains to the DNA ends during ligation and of DNA-PKcs for targeted phosphorylation of other NHEJ proteins as well as trans-phosphorylation of DNA-PKcs on the opposing DSB without disrupting the core ligation complex. Overall the results shed light on evolutionary conservation of Ku, X4, and L4 activities, while explaining the observation that Ku80CTR and DNA-PKcs only occur in a subset of higher eukaryotes.

Toxoplasma gondii peptide ligands open the gate of the HLA class I binding groove.

HLA class I presentation of pathogen-derived peptide ligands is essential for CD8+ T-cell recognition of Toxoplasma gondii infected cells. Currently, little data exist pertaining to peptides that are presented after T. gondii infection. Herein we purify HLA-A*02:01 complexes from T. gondii infected cells and characterize the peptide ligands using LCMS. We identify 195 T. gondii encoded ligands originating from both secreted and cytoplasmic proteins. Surprisingly, T. gondii ligands are significantly longer than uninfected host ligands, and these longer pathogen-derived peptides maintain a canonical N-terminal binding core yet exhibit a C-terminal extension of 1-30 amino acids. Structural analysis demonstrates that binding of extended peptides opens the HLA class I F' pocket, allowing the C-terminal extension to protrude through one end of the binding groove. In summary, we demonstrate that unrealized structural flexibility makes MHC class I receptive to parasite-derived ligands that exhibit unique C-terminal peptide extensions.

Structural Studies of IRF4 Reveal a Flexible Autoinhibitory Region and a Compact Linker Domain.

IRF4 is a unique member of the interferon regulatory factor (IRF) family playing critical regulatory roles in immune cell development, regulation of obesity-induced inflammation, and control of thermogenic gene expression. The ability of IRF4 to control diverse transcriptional programs arises from its proficiency to interact with numerous transcriptional partners. In this study, we present the structural characterization of full-length IRF4. Using a combination of x-ray and small angle x-ray scattering studies, we reveal unique features of the interferon activation domain, including a set of ß-sheets and loops that serve as the binding site for PU.1, and also show that unlike other IRF members, IRF4 has a flexible autoinhibitory region. In addition, we have determined the small angle x-ray scattering solution structure of full-length IRF4, which, together with circular dichroism studies, suggests that the linker region is not extended but folds into a domain structure.

Molecular basis of E. coli L-threonine aldolase catalytic inactivation at low pH.

L-Threonine aldolases (TAs), a family of enzymes belonging to the fold-type I pyridoxal 5'-phosphate (PLP) dependent enzymes, play a role in catalyzing the reversible cleavage of l-3-hydroxy-α-amino acids to glycine and the corresponding aldehydes. Threonine aldolases have great biotechnological potential for the syntheses of pharmaceutically relevant drug molecules because of their stereospecificity. The pH-dependency of their catalytic activity, affecting reaction intermediates, led us to study the effect of low-pH on Escherichia coli TA (eTA) structure. We report here a low-pH crystal structure of eTA at 2.1 Å resolution, with a non-covalently bound uncleaved l-serine substrate, and a PLP cofactor bound as an internal aldimine. This structure contrasts with other eTA structures obtained at physiological pH that show products or substrates bound as PLP-external aldimines. The non-productive binding at low-pH is due to an unusual substrate serine binding orientation in which the α-amino group and carboxylate group are in the wrong positions (relative to the active site residues) as a result of protonation of the α-amino group of the serine, as well as the active site histidines, His83 and His126. Protonation of these residues prevents the characteristic nucleophilic attack of the α-amino group of substrate serine on C4' of PLP to form the external aldimine. Our study shows that at low pH the change in charge distribution at the active site can result in substrates binding in a non-productive orientation.

On the catalytic mechanism and stereospecificity of Escherichia coli L-threonine aldolase.

L-threonine aldolases (L-TAs) represent a family of homologous pyridoxal 5'-phosphate-dependent enzymes found in bacteria and fungi, and catalyse the reversible cleavage of several L-3-hydroxy-α-amino acids. L-TAs have great biotechnological potential, as they catalyse the formation of carbon-carbon bonds, and therefore may be exploited for the bioorganic synthesis of L-3-hydroxyamino acids that are biologically active or constitute building blocks for pharmaceutical molecules. Many L-TAs, showing different stereospecificity towards the Cß configuration, have been isolated. Because of their potential to carry out diastereoselective syntheses, L-TAs have been subjected to structural, functional and mechanistic studies. Nevertheless, their catalytic mechanism and the structural bases of their stereospecificity have not been elucidated. In this study, we have determined the crystal structure of low-specificity L-TA from Escherichia coli at 2.2-Å resolution, in the unliganded form and cocrystallized with L-serine and L-threonine. Furthermore, several active site mutants have been functionally characterized in order to elucidate the reaction mechanism and the molecular bases of stereospecificity. No active site catalytic residue was revealed, and a structural water molecule was assumed to act as the catalytic base in the retro-aldol cleavage reaction. Interestingly, the very large active site opening of E. coli L-TA suggests that much larger molecules than L-threonine isomers may be easily accommodated, and L-TAs may actually have diverse physiological functions in different organisms. Substrate recognition and reaction specificity seem to be guided by the overall microenvironment that surrounds the substrate at the enzyme active site, rather than by one ore more specific residues.