A Selection of Macrocyclic Peptides That Bind STING From an mRNA-Display Library With Split Degenerate Codons.

Recent improvements in mRNA display have enabled the selection of peptides that incorporate non-natural amino acids, thus expanding the chemical diversity of macrocycles beyond what is accessible in nature. Such libraries have incorporated non-natural amino acids at the expense of natural amino acids by reassigning their codons. Here we report an alternative approach to expanded amino-acid diversity that preserves all 19 natural amino acids (no methionine) and adds 6 non-natural amino acids, resulting in the highest sequence complexity reported to date. We have applied mRNA display to this 25-letter library to select functional macrocycles that bind human STING, a protein involved in immunoregulation. The resulting STING-binding peptides include a 9-mer macrocycle with a dissociation constant (KD ) of 3.4 nM, which blocks binding of cGAMP to STING and induces STING dimerization. This approach is generalizable to expanding the amino-acid alphabet in a library beyond 25 building blocks.

High-resolution profiling of MHC II peptide presentation capacity reveals SARS-CoV-2 CD4 T cell targets and mechanisms of immune escape.

Understanding peptide presentation by specific MHC alleles is fundamental for controlling physiological functions of T cells and harnessing them for therapeutic use. However, commonly used in silico predictions and mass spectroscopy have their limitations in precision, sensitivity, and throughput, particularly for MHC class II. Here, we present MEDi, a novel mammalian epitope display that allows an unbiased, affordable, high-resolution mapping of MHC peptide presentation capacity. Our platform provides a detailed picture by testing every antigen-derived peptide and is scalable to all the MHC II alleles. Given the urgent need to understand immune evasion for formulating effective responses to threats such as SARS-CoV-2, we provide a comprehensive analysis of the presentability of all SARS-CoV-2 peptides in the context of several HLA class II alleles. We show that several mutations arising in viral strains expanding globally resulted in reduced peptide presentability by multiple HLA class II alleles, while some increased it, suggesting alteration of MHC II presentation landscapes as a possible immune escape mechanism.

Allelic variation in class I HLA determines CD8+ T cell repertoire shape and cross-reactive memory responses to SARS-CoV-2.

Effective presentation of antigens by human leukocyte antigen (HLA) class I molecules to CD8+ T cells is required for viral elimination and generation of long-term immunological memory. In this study, we applied a single-cell, multiomic technology to generate a unified ex vivo characterization of the CD8+ T cell response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) across four major HLA class I alleles. We found that HLA genotype conditions key features of epitope specificity, TCRα/ß sequence diversity, and the utilization of pre-existing SARS-CoV-2-reactive memory T cell pools. Single-cell transcriptomics revealed functionally diverse T cell phenotypes of SARS-CoV-2-reactive T cells, associated with both disease stage and epitope specificity. Our results show that HLA variations notably influence the CD8+ T cell repertoire shape and utilization of immune recall upon SARS-CoV-2 infection.

An interdomain sector mediating allostery in Hsp70 molecular chaperones.

Allosteric coupling between protein domains is fundamental to many cellular processes. For example, Hsp70 molecular chaperones use ATP binding by their actin-like N-terminal ATPase domain to control substrate interactions in their C-terminal substrate-binding domain, a reaction that is critical for protein folding in cells. Here, we generalize the statistical coupling analysis to simultaneously evaluate co-evolution between protein residues and functional divergence between sequences in protein sub-families. Applying this method in the Hsp70/110 protein family, we identify a sparse but structurally contiguous group of co-evolving residues called a 'sector', which is an attribute of the allosteric Hsp70 sub-family that links the functional sites of the two domains across a specific interdomain interface. Mutagenesis of Escherichia coli DnaK supports the conclusion that this interdomain sector underlies the allosteric coupling in this protein family. The identification of the Hsp70 sector provides a basis for further experiments to understand the mechanism of allostery and introduces the idea that cooperativity between interacting proteins or protein domains can be mediated by shared sectors.

The changing landscape of protein allostery.

It is becoming increasingly clear that the fundamental capacity to undergo conformational change in response to ligand binding is intrinsic to proteins. This property confers on proteins the ability to be allosterically modulated in order to shift substrate binding affinities, alter enzymatic activity or regulate protein-protein interaction. How this allosteric modulation occurs--the pathways of communication, the shifting of conformational ensembles and the altered molecular dynamics--has received considerable attention during the past two years. Recent progress has helped outline the molecular origins of allostery in proteins as diverse as Hsp70 molecular chaperones and signal integrating proteins, such as WASP. In addition, allosteric properties have been successfully engineered into proteins for drug design or the development of novel biosensors. Methodological advances have provided exciting prospects for new insights and new biological roles of allosteric systems have been uncovered.

Computational fluid dynamics simulation of laser-ablated carbon plume propagation in varying background gases for single-walled nanotube synthesis.

A computational fluid dynamics study was conducted to model the plume resulting from the laser ablation of a carbon target in a laser ablation oven for the production of carbon single-walled nanotubes (SWNTs). The goal is to gain understanding into the fluid dynamics and thermodynamics of the plume to ultimately improve SWNT production techniques. The simulations were carried out with a 12-species, 14-reaction chemistry model that included carbon species from C to C6. Metal catalysts in the carbon target were ignored for these simulations. Simulation times ranged from immediate ablation onset to 8 ms past the initial onset of ablation. A secondary goal of the study was to compare computational results with experimental results for three different background gases in the laser ablation oven-argon, helium, and nitrogen. Computational results indicated that lighter carbon species were more quickly diffused into the background gas for helium and nitrogen, resulting in lower localized mass fractions of carbon nanotube "feedstock." The expectation is that this effect will reduce the production of carbon nanotubes, which has been confirmed by experimental evidence. From this investigation, a possible "indicator species" for the production of SWNT appears to be C5.

Hsp70 chaperone ligands control domain association via an allosteric mechanism mediated by the interdomain linker.

Hsp70 chaperones assist in protein folding, disaggregation, and membrane translocation by binding to substrate proteins with an ATP-regulated affinity that relies on allosteric coupling between ATP-binding and substrate-binding domains. We have studied single- and two-domain versions of the E. coli Hsp70, DnaK, to explore the mechanism of interdomain communication. We show that the interdomain linker controls ATPase activity by binding to a hydrophobic cleft between subdomains IA and IIA. Furthermore, the domains of DnaK dock only when ATP binds and behave independently when ADP is bound. Major conformational changes in both domains accompany ATP-induced docking: of particular importance, some regions of the substrate-binding domain are stabilized, while those near the substrate-binding site become destabilized. Thus, the energy of ATP binding is used to form a stable interface between the nucleotide- and substrate-binding domains, which results in destabilization of regions of the latter domain and consequent weaker substrate binding.

Direct comparison of a stable isolated Hsp70 substrate-binding domain in the empty and substrate-bound states.

The Hsp70 family of molecular chaperones acts to prevent protein misfolding, import proteins into organelles, unravel protein aggregates, and enhance cell survival under stress conditions. These activities are all mediated by recognition of diverse hydrophobic sequences via a C-terminal substrate-binding domain. ATP-binding/hydrolysis by the N-terminal ATPase domain regulates the interconversion of the substrate-binding domain between low and high affinity conformations. The empty state of the substrate-binding domain has been difficult to study because of its propensity to bind nearly any available protein chain, even if only modestly hydrophobic. We have generated a new stable construct of the substrate-binding domain from the Escherichia coli Hsp70, DnaK, which has enabled us to compare the empty and peptide-bound conformations using NMR chemical shift analysis and hydrogen-deuterium exchange. We have determined that the empty state is, overall, quite similar to the peptide-bound state, contrary to a previous report. Peptide binding leads to a subtle alteration in the packing of the alpha-helical lid relative to the beta-subdomain. Significantly, we have shown that the chemical shifts of the substrate-binding domain and the ATPase domain do not change when they are placed together in a two-domain construct, whether or not peptide is bound, suggesting that, in the absence of nucleotide, the two domains of E. coli DnaK do not interact. We conclude that the isolated substrate-binding domain exists in a stable high affinity state in the absence of influence from a nucleotide-bound ATPase domain.

Functionally significant mobile regions of Escherichia coli SecA ATPase identified by NMR.

SecA, a 204-kDa homodimeric protein, is a major component of the cellular machinery that mediates the translocation of proteins across the Escherichia coli plasma membrane. SecA promotes translocation by nucleotide-modulated insertion and deinsertion into the cytoplasmic membrane once bound to both the signal sequence and portions of the mature domain of the preprotein. SecA is proposed to undergo major conformational changes during translocation. These conformational changes are accompanied by major rearrangements of SecA structural domains. To understand the interdomain rearrangements, we have examined SecA by NMR and identified regions that display narrow resonances indicating high mobility. The mobile regions of SecA have been assigned to a sequence from the second of two domains with nucleotide-binding folds (NBF-II; residues 564-579) and to the extreme C-terminal segment of SecA (residues 864-901), both of which are essential for preprotein translocation activity. Interactions with ligands suggest that the mobile regions are involved in functionally critical regulatory steps in SecA.