A d-Amino Acid at the N-Terminus of a Protein Abrogates Its Degradation by the N-End Rule Pathway

Eukaryotes have evolved the ubiquitin (Ub)/proteasome system to degrade polypeptides. The Ub/proteasome system is one way that cells regulate cytosolic protein and amino acids levels through the recognition and ubiquitination of a protein’s N-terminus via E1, E2, and E3 enzymes. The process by which the N-terminus stimulates intracellular protein degradation is referred to as the N-end rule. Characterization of the N-end rule has been limited to only the natural l-amino acids. Using a cytosolic delivery platform derived from anthrax lethal toxin, we probed the stability of mixed chirality proteins, containing one d-amino acid on the N-terminus of otherwise all l-proteins. In all cases, we observed that one N-terminal d-amino acid stabilized the cargo protein to proteasomal degradation with respect to the N-end rule. We found that since the mixed chirality proteins were not polyubiquitinated, they evaded N-end-mediated proteasomal degradation. Evidently, a subtle change on the N-terminus of a natural protein can enhance its intracellular lifetime.MIT Faculty Start-up FundMassachusetts Institute of Technology. Charles E. Reed Faculty Initiative FundNational Science Foundation (U.S.) (CAREER Award CHE-1351807)Damon Runyon Cancer Research FoundationNational Science Foundation (U.S.). Graduate Research Fellowship Progra

Translocation of Non-Canonical Polypeptides into Cells Using Protective Antigen

A variety of pathogenic bacteria infect host eukaryotic cells using protein toxins, which enter the cytosol and exert their cytotoxic effects. Anthrax lethal toxin, for example, utilizes the membrane-spanning translocase, protective antigen (PA) pore, to deliver the protein toxin lethal factor (LF) from the endosome into the cytosol of cells. Previous work has investigated the delivery of natural peptides and enzymatic domains appended to the C-terminus of the PA-binding domain of lethal factor (LF[subscript N]) into the cytosol via PA pore. Here, we move beyond natural amino acids and systematically investigate the translocation of polypeptide cargo containing non-canonical amino acids and functionalities through PA pore. Our results indicate translocation is not perturbed with alterations to the peptide backbone or side-chain. Moreover, despite their structural complexity, we found that the small molecule drugs, doxorubicin and monomethyl auristatin F (MMAF) translocated efficiently through PA pore. However, we found cyclic peptides and the small molecule drug docetaxel abrogated translocation due to their large size and structural rigidity. For cargos that reached the cytosol, we demonstrated that each remained intact after translocation. These studies show PA is capable of translocating non-canonical cargo provided it is in a conformational state conducive for passage through the narrow pore.MIT Start-up FundsMassachusetts Institute of Technology. Charles E. Reed Faculty Initiative FundDamon Runyon Cancer Research Foundation (Innovation Award)National Science Foundation (U.S.) (CAREER Award CHE-1351807)National Science Foundation (U.S.). Graduate Research Fellowshi

Rapid Total Synthesis of DARPin pE59 and Barnase

We report the convergent total synthesis of two proteins: DARPin pE59 and Bacillus amyloliquefaciens RNase (Barnase). Leveraging our recently developed fast-flow peptide-synthesis platform, we rapidly explored numerous conditions for the assembly of long polypeptides, and were able to mitigate common side reactions, including deletion and aspartimide products. We report general strategies for improving the synthetic quality of difficult peptide sequences with our system. High-quality protein fragments produced under optimal synthetic conditions were subjected to convergent native chemical ligation, which afforded native full-length proteins after a final desulfurization step. Both DARPin and Barnase were folded and found to be as active as their recombinant analogues.MIT Faculty Start-up FundMassachusetts Institute of Technology (Charles E. Reed Faculty Initiative Fund)Deshpande Center for Technological InnovationDamon Runyon-Rachleff (Innovation Award)Sontag Foundation (Distinguished Scientist Award)AstraZeneca (Firm) (Distinguished Graduate Student Fellowship)Daniel S. Kemp Summer FellowshipNational Institutes of Health (U.S.). Biotechnology Training Program (5T32GM008334-25

Convergent diversity-oriented side-chain macrocyclization scan for unprotected polypeptides

Here we describe a general synthetic platform for side-chain macrocyclization of an unprotected peptide library based on the S[subscript N]Ar reaction between cysteine thiolates and a new generation of highly reactive perfluoroaromatic small molecule linkers. This strategy enabled us to simultaneously “scan” two cysteine residues positioned from i, i + 1 to i, i + 14 sites in a polypeptide, producing 98 macrocyclic products from reactions of 14 peptides with 7 linkers. A complementary reverse strategy was developed; cysteine residues within the polypeptide were first modified with non-bridging perfluoroaryl moieties and then commercially available dithiol linkers were used for macrocyclization. The highly convergent, site-independent, and modular nature of these two strategies coupled with the unique chemoselectivity of a S[subscript N]Ar transformation allows for the rapid diversity-oriented synthesis of hybrid macrocyclic peptide libraries with varied chemical and structural complexities.National Institutes of Health (U.S.) (GM101762)National Institutes of Health (U.S.) (GM046059)MIT Faculty Start-up FundSontag Foundation (Distinguished Scientist Award)Deshpande Center for Technological InnovationMassachusetts Institute of Technology (Charles E. Reed Faculty Initiative Fund)Damon Runyon Cancer Research Foundatio

π-Clamp-mediated cysteine conjugation

Site-selective functionalization of complex molecules is one of the most significant challenges in chemistry. Typically, protecting groups or catalysts must be used to enable the selective modification of one site among many that are similarly reactive, and general strategies that selectively tune the local chemical environment around a target site are rare. Here, we show a four-amino-acid sequence (Phe-Cys-Pro-Phe), which we call the ‘π-clamp’, that tunes the reactivity of its cysteine thiol for site-selective conjugation with perfluoroaromatic reagents. We use the π-clamp to selectively modify one cysteine site in proteins containing multiple endogenous cysteine residues. These examples include antibodies and cysteine-based enzymes that would be difficult to modify selectively using standard cysteine-based methods. Antibodies modified using the π-clamp retained binding affinity to their targets, enabling the synthesis of site-specific antibody–drug conjugates for selective killing of HER2-positive breast cancer cells. The π-clamp is an unexpected approach to mediate site-selective chemistry and provides new avenues to modify biomolecules for research and therapeutics.Massachusetts Institute of Technology (Start-up Funds)National Institutes of Health (U.S.) (NIH R01GM110535)Sontag Foundation (Distinguished Scientist Award)Massachusetts Institute of Technology. Department of Chemistry (George Buchi Research Fellowship)David H. Koch Institute for Integrative Cancer Research at MIT (Graduate Fellowship)Bristol-Myers Squibb Company (Graduate Fellowship in Synthetic Organic Chemistry)National Science Foundation (U.S.) (Graduate Research Fellow

Cholesterol-binding site of the influenza M2 protein in lipid bilayers from solid-state NMR

The influenza M2 protein not only forms a proton channel but also mediates membrane scission in a cholesterol-dependent manner to cause virus budding and release. The atomic interaction of cholesterol with M2, as with most eukaryotic membrane proteins, has long been elusive. We have now determined the cholesterol-binding site of the M2 protein in phospholipid bilayers using solid-state NMR spectroscopy. Chain-fluorinated cholesterol was used to measure cholesterol proximity to M2 while sterol-deuterated cholesterol was used to measure bound-cholesterol orientation in lipid bilayers. Carbon–fluorine distance measurements show that at a cholesterol concentration of 17 mol%, two cholesterol molecules bind each M2 tetramer. Cholesterol binds the C-terminal transmembrane (TM) residues, near an amphipathic helix, without requiring a cholesterol recognition sequence motif. Deuterium NMR spectra indicate that bound cholesterol is approximately parallel to the bilayer normal, with the rough face of the sterol rings apposed to methyl-rich TM residues. The distance- and orientation-restrained cholesterol-binding site structure shows that cholesterol is stabilized by hydrophobic interactions with the TM helix and polar and aromatic interactions with neighboring amphipathic helices. At the 1:2 binding stoichiometry, lipid31P spectra show an isotropic peak indicative of high membrane curvature. This M2–cholesterol complex structure, together with previously observed M2 localization at phase boundaries, suggests that cholesterol mediates M2 clustering to the neck of the budding virus to cause the necessary curvature for membrane scission. The solid-state NMR approach developed here is generally applicable for elucidating the structural basis of cholesterol’s effects on membrane protein function. Keywords: membrane; ¹⁹F-NMR; deuterium NMR; docking; membrane scissio

A structural and mechanistic study of π-clamp-mediated cysteine perfluoroarylation

Natural enzymes use local environments to tune the reactivity of amino acid side chains. In searching for small peptides with similar properties, we discovered a four-residue π-clamp motif (Phe-Cys-Pro-Phe) for regio- and chemoselective arylation of cysteine in ribosomally produced proteins. Here we report mutational, computational, and structural findings directed toward elucidating the molecular factors that drive π-clamp-mediated arylation. We show the significance of a trans conformation prolyl amide bond for the π-clamp reactivity. The π-clamp cysteine arylation reaction enthalpy of activation (ΔH‡) is significantly lower than a non-π-clamp cysteine. Solid-state NMR chemical shifts indicate the prolyl amide bond in the π-clamp motif adopts a 1:1 ratio of the cis and trans conformation, while in the reaction product Pro3 was exclusively in trans. In two structural models of the perfluoroarylated product, distinct interactions at 4.7 Å between Phe1 side chain and perfluoroaryl electrophile moiety are observed. Further, solution 19F NMR and isothermal titration calorimetry measurements suggest interactions between hydrophobic side chains in a π-clamp mutant and the perfluoroaryl probe. These studies led us to design a π-clamp mutant with an 85-fold rate enhancement. These findings will guide us toward the discovery of small reactive peptides to facilitate abiotic chemistry in water.National Institutes of Health (U.S.) (Grant R01GM110535)National Institutes of Health (U.S.) (Grant GM088204)National Science Foundation (U.S.) (Award CHE-1464804

Following Natures Lead: On the Construction of Membrane-Inserted Toxins in Lipid Bilayer Nanodiscs

Bacterial toxin or viral entry into the cell often requires cell surface binding and endocytosis. The endosomal acidification induces a limited unfolding/refolding and membrane insertion reaction of the soluble toxins or viral proteins into their translocation competent or membrane inserted states. At the molecular level, the specific orientation and immobilization of the pre-transitioned toxin on the cell surface is often an important prerequisite prior to cell entry. We propose that structures of some toxin membrane insertion complexes may be observed through procedures where one rationally immobilizes the soluble toxin so that potential unfolding ↔ refolding transitions that occur prior to membrane insertion orientate away from the immobilization surface in the presence of lipid micelle pre-nanodisc structures. As a specific example, the immobilized prepore form of the anthrax toxin pore translocon or protective antigen can be transitioned, inserted into a model lipid membrane (nanodiscs), and released from the immobilized support in its membrane solubilized form. This particular strategy, although unconventional, is a useful procedure for generating pure membrane-inserted toxins in nanodiscs for electron microscopy structural analysis. In addition, generating a similar immobilized platform on label-free biosensor surfaces allows one to observe the kinetics of these acid-induced membrane insertion transitions. These platforms can facilitate the rational design of inhibitors that specifically target the toxin membrane insertion transitions that occur during endosomal acidification. This approach may lead to a new class of direct anti-toxin inhibitors

Monitoring the kinetics of the pH driven transition of the anthrax toxin prepore to the pore by biolayer interferometry and surface plasmon resonance

Domain 2 of the anthrax protective antigen (PA) prepore heptamer unfolds and refolds during endosome acidification to generate an extended 100 Å beta barrel pore that inserts into the endosomal membrane. The PA pore facilitates the pH dependent unfolding and translocation of bound toxin enzymic components, lethal factor (LF) and/or edema factor (EF), from the endosome into the cytoplasm. We constructed immobilized complexes of the prepore with the PA-binding domain of LF (LFN) to monitor the real-time prepore to pore kinetic transition using surface plasmon resonance (SPR) and bio-layer interferometry (BLI). The kinetics of this transition increased as the solution pH was decreased from pH 7.5 to pH 5.0, mirroring acidification of the endosome. Once transitioned, the LFN-PA pore complex was removed from the BLI biosensor tip and deposited onto EM grids, where the PA pore formation was confirmed by negative stain electron microscopy. When the soluble receptor domain (ANTRX2/CMG2) binds the immobilized PA prepore, the transition to the pore state was observed only after the pH was lowered to early or late endosomal pH conditions (5.5 to 5.0 respectively). Once the pore formed, the soluble receptor readily dissociated from the PA pore. Separate binding experiments with immobilized PA pores and soluble receptor indicate that the receptor has a weakened propensity to bind to the transitioned pore. This immobilized anthrax toxin platform can be used to identify or validate potential antimicrobial lead compounds capable of regulating and/or inhibiting anthrax toxin complex formation or pore transitions

Rapid Flow-Based Peptide Synthesis

A flow-based solid-phase peptide synthesis methodology that enables the incorporation of an amino acid residue every 1.8 min under automatic control or every 3 min under manual control is described. This is accomplished by passing a stream of reagent through a heat exchanger into a low volume, low backpressure reaction vessel, and through a UV detector. These features enable continuous delivery of heated solvents and reagents to the solid support at high flow rate, thereby maintaining maximal concentration of reagents in the reaction vessel, quickly exchanging reagents, and eliminating the need to rapidly heat reagents after they have been added to the vessel. The UV detector enables continuous monitoring of the process. To demonstrate the broad applicability and reliability of this method, it was employed in the total synthesis of a small protein, as well as dozens of peptides. The quality of the material obtained with this method is comparable to that for traditional batch methods, and, in all cases, the desired material was readily purifiable by RP-HPLC. The application of this method to the synthesis of the 113-residue Bacillus amyloliquefaciens RNase and the 130-residue DARPin pE59 is described in the accompanying manuscript.MIT Faculty Start-up FundMassachusetts Institute of Technology (Charles E. Reed Faculty Initiative Fund)Deshpande Center for Technological InnovationDamon Runyon-Rachleff (Innovation Award)Sontag Foundation (Distinguished Scientist Award)C. P. Chu and Y. Lai FellowshipDaniel S. Kemp Summer FellowshipNational Institute of General Medical Sciences (U.S.). Biotechnology Training Program (Grant 5T32GM008334-25)National Institutes of Health (U.S.) (Fellowship F32GM101762