28Coronavirus disease 19 is an emerging global health crisis. With over 200,000 29 confirmed cases to date, this pandemic continues to expand, spurring research to discover 30 vaccines and therapies. SARS-CoV-2 is the novel coronavirus responsible for this disease. It 31 initiates entry into human cells by binding to angiotensin-converting enzyme 2 (ACE2) via the 32 receptor binding domain (RBD) of its spike protein (S). Disrupting the SARS-CoV-2-RBD binding 33 to ACE2 with designer drugs has the potential to inhibit the virus from entering human cells, 34 presenting a new modality for therapeutic intervention. Peptide-based binders are an attractive 35 solution to inhibit the RBD-ACE2 interaction by adequately covering the extended protein contact 36interface. Using molecular dynamics simulations based on the recently solved ACE2 and SARS-37CoV-2-RBD co-crystal structure, we observed that the ACE2 peptidase domain (PD) α1 helix is 38 important for binding SARS-CoV-2-RBD. Using automated fast-flow peptide synthesis, we 39 chemically synthesized a 23-mer peptide fragment of the ACE2 PD α1 helix composed entirely of 40 proteinogenic amino acids. Chemical synthesis of this human derived sequence was complete in 41 1.5 hours and after work up and isolation >20 milligrams of pure material was obtained. Bio-layer 42 interferometry revealed that this peptide specifically associates with the SARS-CoV-2-RBD with 43 low nanomolar affinity. This peptide binder to SARS-CoV-2-RBD provides new avenues for 44 COVID-19 treatment and diagnostic modalities by blocking the SARS-CoV-2 spike protein 45 interaction with ACE2 and thus precluding virus entry into human cells. 46 47 Key words ： SARS-CoV-2, peptide binder, protein-protein interaction inhibitor, coronavirus, 48 COVID-19, rapid response, peptide therapeutic, MD simulation, automated flow peptide synthesis 49 50 51 3

The influenza B M2 (BM2) proton channel is activated by acidic pH to mediate virus uncoating. Unlike influenza A M2 (AM2), which conducts protons with strong inward rectification, BM2 conducts protons both inward and outward. Here we report 1.4- and 1.5-angstrom solid-state NMR structures of the transmembrane domain of the closed and open BM2 channels in phospholipid environment. Upon activation, the transmembrane helices increase the tilt angle by 6˚ and the average pore diameter enlarges by 2.1 Å. BM2 thus undergoes a scissor motion for activation, which differs from the alternating-access motion of AM2. These results indicate that asymmetric proton conduction requires a backbone hinge motion whereas bidirectional conduction is achieved by a symmetric scissor motion. The proton-selective histidine and gating tryptophan in the open BM2 reorient on the microsecond timescale, similar to AM2, indicating that sidechain dynamics are the essential driver of proton shuttling.

Few chemical methods exist for the covalent conjugation of two proteins. We report the preparation of site-specific protein–protein conjugates that arise from the sequential cross-coupling of cysteine residues on two different proteins. The method involves the synthesis of stable palladium–protein oxidative addition complexes (Pd-protein OACs), a process that converts nucleo­philic cysteine residues into an electro­philic S-aryl-Pd-X unit by taking advantage of an intra­molecular oxidative addition strategy. This process is demonstrated on proteins up to 83 kDa in size and can be conveniently carried out in water and open to air. The resulting Pd-protein OACs can cross-couple with other thiol-containing proteins to arrive at homogeneous protein–protein bioconjugates.

Pseudomonas aeruginosa is a major bacterial pathogen associated with a rising prevalence of antibiotic resistance. We evaluated the resistance mechanisms of P. aeruginosa against POL7080, a species-specific, first-in-class antibiotic in clinical trials that targets the lipopolysaccharide transport protein LptD. We isolated a series of POL7080-resistant strains with mutations in the two-component sensor gene pmrB. Transcriptomic and confocal microscopy studies support a resistance mechanism shared with colistin, involving lipopolysaccharide modifications that mitigate antibiotic cell surface binding.

The nontoxic, anthrax protective antigen/lethal factor N‐terminal domain (PA/LFN) complex is an effective platform for translocating proteins into the cytosol of cells. Mutant PA (mPA) was recently fused to epidermal growth factor (EGF) to retarget delivery of LFN to cells bearing EGF receptors (EGFR), but the requirement for a known cognate ligand limits the applicability of this approach. Here, we render practical protective antigen retargeting to a variety of receptors with mPA single‐chain variable fragment (scFv) fusion constructs. Our design enables the targeting of two pancreatic cancer‐relevant receptors, EGFR and carcinoembryonic antigen. We demonstrate that fusion to scFvs does not disturb the basic functions of mPA. Moreover, mPA−scFv fusions enable cell‐specific delivery of diphtheria toxin catalytic domain and Ras/Rap1‐specific endopeptidase to pancreatic cancer cells. Importantly, mPA−scFv fusion‐based treatments display potent cell‐specific toxicity in vitro, opening fundamentally new routes toward engineered immunotoxins and providing a potential solution to the challenge of targeted protein delivery to the cytosol of cancer cells.