Safeguarding intestine cells against enteropathogenic Escherichia coli by intracellular protein reaction, a preventive antibacterial mechanism.

A critical problem in the fight against bacterial infection is the rising rates of resistance and the lack of new antibiotics. The discovery of new targets or new antibacterial mechanisms is a potential solution but is becoming more difficult. Here we report an antibacterial mechanism that safeguards intestine cells from enteropathogenic Escherichia coli (EPEC) by shutting down an infection-responsive signal of the host intestine cell. A key step in EPEC infection of intestinal cells involves Tir-induced actin reorganization. Nck mediates this event by binding with Tir through its SH2 domain (Nck-SH2) and with WIP through its second SH3 domain (Nck-SH3.2). Here we report the design of a synthetic peptide that reacts precisely with a unique cysteine of the Nck-SH3.2 domain, blocks the binding site of the Nck protein, and prevents EPEC infection of Caco-2 cells. Oral update of this nontoxic peptide before EPEC administration safeguards mice from EPEC infection and diarrhea. This study demonstrates domain-specific blockage of an SH3 domain of a multidomain adaptor protein inside cells and the inhibition of Tir-induced rearrangement of the host actin cytoskeleton as a previously unknown antibacterial mechanism.

Targeted Covalent Inhibition of Grb2-Sos1 Interaction through Proximity-Induced Conjugation in Breast Cancer Cells.

Targeted covalent inhibitors of protein-protein interactions differ from reversible inhibitors in that the former bind and covalently bond the target protein at a specific site of the target. The site specificity is the result of the proximity of two reactive groups at the bound state, for example, one mild electrophile in the inhibitor and a natural cysteine in the target close to the ligand binding site. Only a few pharmaceutically relevant proteins have this structural feature. Grb2, a key adaptor protein in maintaining the ERK activity via binding Sos1 to activated RTKs, is one: the N-terminal SH3 domain of Grb2 (Grb2N-SH3) carries a unique solvent-accessible cysteine Cys32 close to its Sos1-binding site. Here we report the design of a peptide-based antagonist (a reactive peptide) that specifically binds to Grb2N-SH3 and subsequently undergoes a nucleophilic reaction with Cys32 to form a covalent bond thioether, to block Grb2-Sos1 interaction. Through rounds of optimization, we eventually obtained a dimeric reaction reactive peptide that can form a covalent adduct with endogenous Grb2 protein inside the cytosol of SK-BR-3 human breast cancer cells with pronounced inhibitory effect on cell mobility and viability. This work showcases a rational design of Grb2-targeted site-specific covalent inhibitor and its pronounced anticancer effect by targeting Grb2-Sos1 interaction.

Site Selective Azo Coupling for Peptide Cyclization and Affinity Labeling of an SH3 Protein.

A key challenge in bioconjugation is to control the site selectivity of the reaction. Chemical reagents often react with proteineous chemical groups without showing preference to their location or microenvironment in the protein; to confine the reaction to an amino acid at a specific site, one needs to distinguish this residue from others despite their identical chemical properties. Here, we report a strategy that utilizes proximity-driven reactivity to achieve site selective azo coupling between tyrosine and aryldiazonium. A phenylalanine analogue with an aryldiazonium moiety at its side chain was incorporated into a synthetic peptide and was found to react only with tyrosine in its vicinity but also to remain inert to others that are not immediately adjacent, a property attained by fine regulation of the electronic effect of the substituent on the aryl ring. Proximity-driven intramolecular azo coupling was showcased in cyclization of a ß-hairpin peptide, structural features of the azo linked cyclic peptide was elucidated by NMR, and intermolecular azo coupling was achieved between an SH3 protein Abl-SH3 and its polyproline peptide ligands at specific tyrosine residues. This approach is generally applicable to develop covalent affinity labels for SH3 proteins because of the high occurrence rate of tyrosine at the peptide-binding site of SH3 proteins.

Cascade biocatalysis by multienzyme-nanoparticle assemblies.

Multienzyme complexes are of paramount importance in biosynthesis in cells. Yet, how sequential enzymes of cascade catalytic reactions synergize their activities through spatial organization remains elusive. Recent development of site-specific protein-nanoparticle conjugation techniques enables us to construct multienzyme assemblies using nanoparticles as the template. Sequential enzymes in menaquinone biosynthetic pathway were conjugated to CdSe-ZnS quantum dots (QDs, a nanosized particulate material) through metal-affinity driven self-assembly. The assemblies were characterized by electrophoretic methods, the catalytic activities were monitored by reverse-phase chromatography, and the composition of the multienzyme-QD assemblies was optimized through a progressive approach to achieve highly efficient catalytic conversion. Shorter enzyme-enzyme distance was discovered to facilitate intermediate transfer, and a fine control on the stoichiometric ratio of the assembly was found to be critical for the maximal synergy between the enzymes. Multienzyme-QD assemblies thereby provide an effective model to scrutinize the synergy of cascade enzymes in multienzyme complexes.

Assembly of multivalent protein ligands and quantum dots: a multifaceted investigation.

The development of multivalent protein ligands for nanoparticles lags behind that of multidentate polymers and small-molecule ligands largely because of a lack of thorough understanding of the interaction between nanoparticles and multimeric proteins. Guided by protein crystal structures, we have harnessed recombinant technology to develop a collection of mCherry fused multimeric proteins with different spatial distributions of the quantum dot (QD)-binding sequence, hexahistidine tag (histag). All of the proteins can behave as ligands to assemble with ZnS-CdSe QDs through metal-affinity-driven self-assembly. We have observed that protein shape and geometry greatly affect the stoichiometry and stability of their assemblies with QDs. We also demonstrate a peptide-induced structural transition of a nanobelt protein that preorganizes the QD-binding sites and effects a more efficient assembly with QDs. This work reports the first multifaceted investigation on how multivalent proteins, in particular, dimers, tetramers, and linear multidentate proteins, assemble with QDs. It also manifests our capability of harnessing structural and conformational information about proteins to design multivalent protein ligands for QD surface functionalization.

PDZ-Reactive Peptide Activates Ephrin-B Reverse Signaling and Inhibits Neuronal Chemotaxis.

Intracellular reactions on nonenzymatic proteins that activate cellular signals are rarely found. We report one example here that a designed peptide derivative undergoes a nucleophilic reaction specifically with a cytosolic PDZ protein inside cells. This reaction led to the activation of ephrin-B reverse signaling, which subsequently inhibited SDF-1 induced neuronal chemotaxis of human neuroblastoma cells and mouse cerebellar granule neurons. Our work provides direct evidence that PDZ-RGS3 bridges ephrin-B reverse signaling and SDF-1 induced G protein signaling for the first time.