Engineering dynamical control of cell fate switching using synthetic phospho-regulons.

Many cells can sense and respond to time-varying stimuli, selectively triggering changes in cell fate only in response to inputs of a particular duration or frequency. A common motif in dynamically controlled cells is a dual-timescale regulatory network: although long-term fate decisions are ultimately controlled by a slow-timescale switch (e.g., gene expression), input signals are first processed by a fast-timescale signaling layer, which is hypothesized to filter what dynamic information is efficiently relayed downstream. Directly testing the design principles of how dual-timescale circuits control dynamic sensing, however, has been challenging, because most synthetic biology methods have focused solely on rewiring transcriptional circuits, which operate at a single slow timescale. Here, we report the development of a modular approach for flexibly engineering phosphorylation circuits using designed phospho-regulon motifs. By then linking rapid phospho-feedback with slower downstream transcription-based bistable switches, we can construct synthetic dual-timescale circuits in yeast in which the triggering dynamics and the end-state properties of the ON state can be selectively tuned. These phospho-regulon tools thus open up the possibility to engineer cells with customized dynamical control.

A Four-Monoclonal Antibody Combination Potently Neutralizes Multiple Botulinum Neurotoxin Serotypes C and D.

Human botulism can be caused by botulinum neurotoxin (BoNT) serotypes A to G. Here, we present an antibody-based antitoxin composed of four human monoclonal antibodies (mAbs) against BoNT/C, BoNT/D, and their mosaic toxins. This work built on our success in generating protective mAbs to BoNT /A, B and E serotypes. We generated mAbs from human immune single-chain Fv (scFv) yeast-display libraries and isolated scFvs with high affinity for BoNT/C, BoNT/CD, BoNT/DC and BoNT/D serotypes. We identified four mAbs that bound non-overlapping epitopes on multiple serotypes and mosaic BoNTs. Three of the mAbs underwent molecular evolution to increase affinity. A four-mAb combination provided high-affinity binding and BoNT neutralization of both serotypes and their mosaic toxins. The mAbs have potential utility as therapeutics and as diagnostics capable of recognizing and neutralizing BoNT/C and BoNT/D serotypes and their mosaic toxins. A derivative of the four-antibody combination (NTM-1634) completed a Phase 1 clinical trial (Snow et al., Antimicrobial Agents and Chemotherapy, 2019) with no drug-related serious adverse events.

Hydrophobic surface burial is the major stability determinant of a flat, single-layer beta-sheet.

Formation of a flat beta-sheet is a fundamental event in beta-sheet-mediated protein self-assembly. To investigate the contributions of various factors to the stability of flat beta-sheets, we performed extensive alanine-scanning mutagenesis experiments on the single-layer beta-sheet segment of Borrelia outer surface protein A (OspA). This beta-sheet segment consists of beta-strands with highly regular geometries that can serve as a building block for self-assembly. Our Ala-scanning approach is distinct from the conventional host-guest method, in that it introduces only conservative, truncation mutations that should minimize structural perturbation. Our results showed very weak correlation with experimental beta-sheet propensity scales, statistical beta-sheet propensity scales, or cross-strand pairwise correlations. In contrast, our data showed strong positive correlation with the change in buried non-polar surface area. Polar interactions including prominent Glu-Lys cross-strand pairs contribute marginally to the beta-sheet stability. These results were corroborated by results from additional non-Ala mutations. Taken together, these results demonstrate the dominant contribution of non-polar surface burial to flat beta-sheet stability even at solvent-exposed positions. The OspA single-layer beta-sheet achieves efficient hydrophobic surface burial without forming a hydrophobic core by a strategic placement of a variety of side-chains. These findings further suggest the importance of hydrophobic interactions within a beta-sheet layer in peptide self-assembly.

Beta-strand flipping and slipping triggered by turn replacement reveal the opportunistic nature of beta-strand pairing.

We investigated how the register between adjacent beta-strands is specified using a series of mutants of the single-layer beta-sheet (SLB) in Borrelia OspA. The single-layer architecture of this system eliminates structural restraints imposed by a hydrophobic core, enabling us to address this question. A critical turn (turn 9/10) in the SLB was replaced with a segment with an intentional structural mismatch. Its crystal structure revealed a one-residue insertion into the central beta-strand (strand 9) of the SLB. This insertion triggered a surprisingly large-scale structural rearrangement: (i) the central strand (strand 9) was shifted by one residue, causing the strand to flip with respect to the adjacent beta-strands and thus completely disrupting the native side-chain contacts; (ii) the three-residue turn located on the opposite end of the beta-strand (turn 8/9) was pushed into its preceding beta-strand (strand 8); (iii) the register between strands 8 and 9 was shifted by three residues. Replacing the original sequence for turn 8/9 with a stronger turn motif restored the original strand register but still with a flipped beta-strand 9. The stability differences of these distinct structures were surprisingly small, consistent with an energy landscape where multiple low-energy states with different beta-sheet configurations exist. The observed conformations can be rationalized in terms of maximizing the number of backbone H-bonds. These results suggest that adjacent beta-strands "stick" through the use of factors that are not highly sequence specific and that beta-strands could slide back and forth relatively easily in the absence of external elements such as turns and tertiary packing.

Atomic-resolution crystal structure of Borrelia burgdorferi outer surface protein A via surface engineering.

Outer surface protein A (OspA) from Borrelia burgdorferi has an unusual dumbbell-shaped structure in which two globular domains are connected with a "single-layer" beta-sheet (SLB). The protein is highly soluble, and it has been recalcitrant to crystallization. Only OspA complexes with Fab fragments have been successfully crystallized. OspA contains a large number of Lys and Glu residues, and these "high entropy" residues may disfavor crystal packing because some of them would need to be immobilized in forming a crystal lattice. We rationally designed a total of 13 surface mutations in which Lys and Glu residues were replaced with Ala or Ser. We successfully crystallized the mutant OspA without a bound Fab fragment and extended structure analysis to a 1.15 Angstroms resolution. The new high-resolution structure revealed a unique backbone hydration pattern of the SLB segment in which water molecules fill the "weak spots" on both faces of the antiparallel beta-sheet. These well-defined water molecules provide additional structural links between adjacent beta-strands, and thus they may be important for maintaining the rigidity of the SLB that inherently lacks tight packing afforded by a hydrophobic core. The structure also revealed new information on the side-chain dynamics and on a solvent-accessible cavity in the core of the C-terminal globular domain. This work demonstrates the utility of extensive surface mutation in crystallizing recalcitrant proteins and dramatically improving the resolution of crystal structures, and provides new insights into the stabilization mechanism of OspA.

Thermodynamic and kinetic exploration of the energy landscape of Borrelia burgdorferi OspA by native-state hydrogen exchange.

We report a native-state hydrogen-exchange (HX) method to simultaneously obtain both thermodynamic and kinetic information on the formation of multiple excited states in a folding energy landscape. Our method exploits the inherent dispersion and pH dependence of the intrinsic HX rates to cover both the EX2 (thermodynamic) and EX1 (kinetic) regimes. At each concentration of denaturant, HX measurements are performed over a range of pH values. Using this strategy, we dissected Borrelia burgdorferi OspA, a predominantly beta-sheet protein containing a unique single-layer beta-sheet, into five cooperative units and postulated excited states predominantly responsible for HX. More importantly, we determined the interconversion rates between these excited states and the native state. The use of both thermodynamic and kinetic information from native-state HX enabled us to construct a folding landscape of this 28kDa protein, including local minima and maxima, and to discriminate on-pathway and off-pathway intermediates. This method, which we term EX2/EX1 HX, should be a powerful tool for characterizing the complex folding mechanisms exhibited by the majority of proteins.

Conformational heterogeneity of an equilibrium folding intermediate quantified and mapped by scanning mutagenesis.

It is challenging to experimentally define an energy landscape for protein folding that comprises multiple partially unfolded states. Experimental results are often ambiguous as to whether a non-native state is conformationally homogeneous. Here, we tested an approach combining systematic mutagenesis and a Brønsted-like analysis to reveal and quantify conformational heterogeneity of folding intermediate states. Using this method, we resolved an otherwise apparently homogeneous equilibrium folding intermediate of Borrelia burgdorferi OspA into two conformationally distinct species and determined their relative populations. Furthermore, we mapped the structural differences between these intermediate species, which are consistent with the non-native species that we previously proposed based on native-state hydrogen exchange studies. When treated as a single state, the intermediate ensemble exhibited fractional Phi-values for mutations and Hammond-type behaviors that are often observed for folding transition states. We found that a change in relative population of the two species within the intermediate ensemble explains these properties well, suggesting that fractional Phi-values and Hammond-type behaviors exhibited by folding intermediates and transition states may arise more often from conformational heterogeneity than from a single partial structure. Our results are consistent with the presence of multiple minima in a rugged energy landscape predicted from theoretical studies. The method described here provides a promising means to probe a complex folding energy landscape.

Aromatic cluster mutations produce focal modulations of ß-sheet structure.

Site-directed mutagenesis is a powerful tool for altering the structure and function of proteins in a focused manner. Here, we examined how a model ß-sheet protein could be tuned by mutation of numerous surface-exposed residues to aromatic amino acids. We designed these aromatic side chain "clusters" at highly solvent-exposed positions in the flat, single-layer ß-sheet of Borrelia outer surface protein A (OspA). This unusual ß-sheet scaffold allows us to interrogate the effects of these mutations in the context of well-defined structure but in the absence of the strong scaffolding effects of globular protein architecture. We anticipated that the introduction of a cluster of aromatic amino acid residues on the ß-sheet surface would result in large conformational changes and/or stabilization and thereby provide new means of controlling the properties of ß-sheets. Surprisingly, X-ray crystal structures revealed that the introduction of aromatic clusters produced only subtle conformational changes in the OspA ß-sheet. Additionally, despite burying a large degree of hydrophobic surface area, the aromatic cluster mutants were slightly less stable than the wild-type scaffold. These results thereby demonstrate that the introduction of aromatic cluster mutations can serve as a means for subtly modulating ß-sheet conformation in protein design.

Using engineered scaffold interactions to reshape MAP kinase pathway signaling dynamics.

Scaffold proteins link signaling molecules into linear pathways by physically assembling them into complexes. Scaffolds may also have a higher-order role as signal-processing hubs, serving as the target of feedback loops that optimize signaling amplitude and timing. We demonstrate that the Ste5 scaffold protein can be used as a platform to systematically reshape output of the yeast mating MAP kinase pathway. We constructed synthetic positive- and negative-feedback loops by dynamically regulating recruitment of pathway modulators to an artificial binding site on Ste5. These engineered circuits yielded diverse behaviors: ultrasensitive dose response, accelerated or delayed response times, and tunable adaptation. Protein scaffolds provide a flexible platform for reprogramming cellular responses and could be exploited to engineer cells with novel therapeutic and biotechnological functions.

High-resolution structure of a self-assembly-competent form of a hydrophobic peptide captured in a soluble beta-sheet scaffold.

beta-Rich self-assembly is a major structural class of polypeptides, but still little is known about its atomic structures and biophysical properties. Major impediments for structural and biophysical studies of peptide self-assemblies include their insolubility and heterogeneous composition. We have developed a model system, termed peptide self-assembly mimic (PSAM), based on the single-layer beta-sheet of Borrelia outer surface protein A. PSAM allows for the capture of a defined number of self-assembly-like peptide repeats within a water-soluble protein, making structural and energetic studies possible. In this work, we extend our PSAM approach to a highly hydrophobic peptide sequence. We show that a penta-Ile peptide (Ile(5)), which is insoluble and forms beta-rich self-assemblies in aqueous solution, can be captured within the PSAM scaffold in a form capable of self-assembly. The 1.1-A crystal structure revealed that the Ile(5) stretch forms a highly regular beta-strand within this flat beta-sheet. Self-assembly models built with multiple copies of the crystal structure of the Ile(5) peptide segment showed no steric conflict, indicating that this conformation represents an assembly-competent form. The PSAM retained high conformational stability, suggesting that the flat beta-strand of the Ile(5) stretch primed for self-assembly is a low-energy conformation of the Ile(5) stretch and rationalizing its high propensity for self-assembly. The ability of the PSAM to "solubilize" an otherwise insoluble peptide stretch suggests the potential of the PSAM approach to the characterization of self-assembling peptides.

Atomic structures of peptide self-assembly mimics.

Although the beta-rich self-assemblies are a major structural class for polypeptides and the focus of intense research, little is known about their atomic structures and dynamics due to their insoluble and noncrystalline nature. We developed a protein engineering strategy that captures a self-assembly segment in a water-soluble molecule. A predefined number of self-assembling peptide units are linked, and the beta-sheet ends are capped to prevent aggregation, which yields a mono-dispersed soluble protein. We tested this strategy by using Borrelia outer surface protein (OspA) whose single-layer beta-sheet located between two globular domains consists of two beta-hairpin units and thus can be considered as a prototype of self-assembly. We constructed self-assembly mimics of different sizes and determined their atomic structures using x-ray crystallography and NMR spectroscopy. Highly regular beta-sheet geometries were maintained in these structures, and peptide units had a nearly identical conformation, supporting the concept that a peptide in the regular beta-geometry is primed for self-assembly. However, we found small but significant differences in the relative orientation between adjacent peptide units in terms of beta-sheet twist and bend, suggesting their inherent flexibility. Modeling shows how this conformational diversity, when propagated over a large number of peptide units, can lead to a substantial degree of nanoscale polymorphism of self-assemblies.