One-shot design elevates functional expression levels of a voltage-gated potassium channel.

Membrane proteins play critical physiological roles as receptors, channels, pumps, and transporters. Despite their importance, however, low expression levels often hamper the experimental characterization of membrane proteins. We present an automated and web-accessible design algorithm called mPROSS (https://mPROSS.weizmann.ac.il), which uses phylogenetic analysis and an atomistic potential, including an empirical lipophilicity scale, to improve native-state energy. As a stringent test, we apply mPROSS to the Kv1.2-Kv2.1 paddle chimera voltage-gated potassium channel. Four designs, encoding 9-26 mutations relative to the parental channel, were functional and maintained potassium-selective permeation and voltage dependence in Xenopus oocytes with up to 14-fold increase in whole-cell current densities. Additionally, single-channel recordings reveal no significant change in the channel-opening probability nor in unitary conductance, indicating that functional expression levels increase without impacting the activity profile of individual channels. Our results suggest that the expression levels of other dynamic channels and receptors may be enhanced through one-shot design calculations.

Design of a stable human acid-ß-glucosidase: towards improved Gaucher disease therapy and mutation classification.

Acid-ß-glucosidase (GCase, EC3.2.1.45), the lysosomal enzyme which hydrolyzes the simple glycosphingolipid, glucosylceramide (GlcCer), is encoded by the GBA1 gene. Biallelic mutations in GBA1 cause the human inherited metabolic disorder, Gaucher disease (GD), in which GlcCer accumulates, while heterozygous GBA1 mutations are the highest genetic risk factor for Parkinson's disease (PD). Recombinant GCase (e.g., Cerezyme® ) is produced for use in enzyme replacement therapy for GD and is largely successful in relieving disease symptoms, except for the neurological symptoms observed in a subset of patients. As a first step toward developing an alternative to the recombinant human enzymes used to treat GD, we applied the PROSS stability-design algorithm to generate GCase variants with enhanced stability. One of the designs, containing 55 mutations compared to wild-type human GCase, exhibits improved secretion and thermal stability. Furthermore, the design has higher enzymatic activity than the clinically used human enzyme when incorporated into an AAV vector, resulting in a larger decrease in the accumulation of lipid substrates in cultured cells. Based on stability-design calculations, we also developed a machine learning-based approach to distinguish benign from deleterious (i.e., disease-causing) GBA1 mutations. This approach gave remarkably accurate predictions of the enzymatic activity of single-nucleotide polymorphisms in the GBA1 gene that are not currently associated with GD or PD. This latter approach could be applied to other diseases to determine risk factors in patients carrying rare mutations.

CDR1 Composition Can Affect Nanobody Recombinant Expression Yields.

The isolation of nanobodies from pre-immune libraries by means of biopanning is a straightforward process. Nevertheless, the recovered candidates often require optimization to improve some of their biophysical characteristics. In principle, CDRs are not mutated because they are likely to be part of the antibody paratope, but in this work, we describe a mutagenesis strategy that specifically addresses CDR1. Its sequence was identified as an instability hot spot by the PROSS program, and the available structural information indicated that four CDR1 residues bound directly to the antigen. We therefore modified the loop flexibility with the addition of an extra glycine rather than by mutating single amino acids. This approach significantly increased the nanobody yields but traded-off with moderate affinity loss. Accurate modeling coupled with atomistic molecular dynamics simulations enabled the modifications induced by the glycine insertion and the rationale behind the engineering design to be described in detail.

Local Mutations Can Serve as a Game Changer for Global Protein Solvent Interaction.

Although it is well-known that limited local mutations of enzymes, such as matrix metalloproteinases (MMPs), may change enzyme activity by orders of magnitude as well as its stability, the completely rational design of proteins is still challenging. These local changes alter the electrostatic potential and thus local electrostatic fields, which impacts the dynamics of water molecules close the protein surface. Here we show by a combined computational design, experimental, and molecular dynamics (MD) study that local mutations have not only a local but also a global effect on the solvent: In the specific case of the matrix metalloprotease MMP14, we found that the nature of local mutations, coupled with surface morphology, have the ability to influence large patches of the water hydrogen-bonding network at the protein surface, which is correlated with stability. The solvent contribution can be experimentally probed via terahertz (THz) spectroscopy, thus opening the door to the exciting perspective of rational protein design in which a systematic tuning of hydration water properties allows manipulation of protein stability and enzymatic activity.

Community-Wide Experimental Evaluation of the PROSS Stability-Design Method.

Recent years have seen a dramatic improvement in protein-design methodology. Nevertheless, most methods demand expert intervention, limiting their widespread adoption. By contrast, the PROSS algorithm for improving protein stability and heterologous expression levels has been successfully applied to a range of challenging enzymes and binding proteins. Here, we benchmark the application of PROSS as a stand-alone tool for protein scientists with no or limited experience in modeling. Twelve laboratories from the Protein Production and Purification Partnership in Europe (P4EU) challenged the PROSS algorithm with 14 unrelated protein targets without support from the PROSS developers. For each target, up to six designs were evaluated for expression levels and in some cases, for thermal stability and activity. In nine targets, designs exhibited increased heterologous expression levels either in prokaryotic and/or eukaryotic expression systems under experimental conditions that were tailored for each target protein. Furthermore, we observed increased thermal stability in nine of ten tested targets. In two prime examples, the human Stem Cell Factor (hSCF) and human Cadherin-Like Domain (CLD12) from the RET receptor, the wild type proteins were not expressible as soluble proteins in E. coli, yet the PROSS designs exhibited high expression levels in E. coli and HEK293 cells, respectively, and improved thermal stability. We conclude that PROSS may improve stability and expressibility in diverse cases, and that improvement typically requires target-specific expression conditions. This study demonstrates the strengths of community-wide efforts to probe the generality of new methods and recommends areas for future research to advance practically useful algorithms for protein science.

PROSS 2: a new server for the design of stable and highly expressed protein variants.

SUMMARY: Many natural and designed proteins are only marginally stable limiting their usefulness in research and applications. Recently, we described an automated structure and sequence-based design method, called PROSS, for optimizing protein stability and heterologous expression levels that has since been validated on dozens of proteins. Here, we introduce improvements to the method, workflow and presentation, including more accurate sequence analysis, error handling and automated analysis of the quality of the sequence alignment that is used in design calculations. AVAILABILITY AND IMPLEMENTATION: PROSS2 is freely available for academic use at https://pross.weizmann.ac.il.

One-step sequence and structure-guided optimization of HIV-1 envelope gp140.

Stabilization of the metastable envelope glycoprotein (Env) of HIV-1 is hypothesized to improve induction of broadly neutralizing antibodies. We improved the expression yield and stability of the HIV-1 envelope glycoprotein BG505SOSIP.664 gp140 by means of a previously described automated sequence and structure-guided computational thermostabilization approach, PROSS. This combines sequence conservation information with computational assessment of mutant stabilization, thus taking advantage of the extensive natural sequence variation present in HIV-1 Env. PROSS is used to design three gp140 variants with 17-45 mutations relative to the parental construct. One of the designs is experimentally observed to have a fourfold improvement in yield and a 4 °C increment in thermostability. In addition, the designed immunogens have similar antigenicity profiles to the native flexible linker version of wild type, BG505SOSIP.664 gp140 (NFL Wt) to major epitopes targeted by broadly neutralizing antibodies. PROSS eliminates the laborious process of screening many variants for stability and functionality, providing a proof of principle of the method for stabilization and improvement of yield without compromising antigenicity for next generation complex, highly glycosylated vaccine candidates.

Manipulating the Folding Landscape of a Multidomain Protein.

Folding of proteins to their functional conformation is paramount to life. Though 75% of the proteome consists of multidomain proteins, our knowledge of folding has been based primarily on studies conducted on single-domain and fast-folding proteins. Nonetheless, the complexity of folding landscapes exhibited by multidomain proteins has received increased scrutiny in recent years. We study the three-domain protein adenylate kinase from E. coli (AK), which has been shown to fold through a series of pathways involving several intermediate states. We use a protein design method to manipulate the folding landscape of AK, and single-molecule FRET spectroscopy to study the effects on the folding process. Mutations introduced in the NMP binding (NMPbind) domain of the protein are found to have unexpected effects on the folding landscape. Thus, while stabilizing mutations in the core of the NMPbind domain retain the main folding pathways of wild-type AK, a destabilizing mutation at the interface between the NMPbind and the CORE domains causes a significant repartition of the flux between the folding pathways. Our results demonstrate the outstanding plasticity of the folding landscape of AK and reveal how specific mutations in the primary structure are translated into changes in folding dynamics. The combination of methodologies introduced in this work should prove useful for deepening our understanding of the folding process of multidomain proteins.

Principles of Protein Stability and Their Application in Computational Design.

Proteins are increasingly used in basic and applied biomedical research. Many proteins, however, are only marginally stable and can be expressed in limited amounts, thus hampering research and applications. Research has revealed the thermodynamic, cellular, and evolutionary principles and mechanisms that underlie marginal stability. With this growing understanding, computational stability design methods have advanced over the past two decades starting from methods that selectively addressed only some aspects of marginal stability. Current methods are more general and, by combining phylogenetic analysis with atomistic design, have shown drastic improvements in solubility, thermal stability, and aggregation resistance while maintaining the protein's primary molecular activity. Stability design is opening the way to rational engineering of improved enzymes, therapeutics, and vaccines and to the application of protein design methodology to large proteins and molecular activities that have proven challenging in the past.

Local energetic frustration affects the dependence of green fluorescent protein folding on the chaperonin GroEL.

The GroE chaperonin system in Escherichia coli comprises GroEL and GroES and facilitates ATP-dependent protein folding in vivo and in vitro Proteins with very similar sequences and structures can differ in their dependence on GroEL for efficient folding. One potential but unverified source for GroEL dependence is frustration, wherein not all interactions in the native state are optimized energetically, thereby potentiating slow folding and misfolding. Here, we chose enhanced green fluorescent protein as a model system and subjected it to random mutagenesis, followed by screening for variants whose in vivo folding displays increased or decreased GroEL dependence. We confirmed the altered GroEL dependence of these variants with in vitro folding assays. Strikingly, mutations at positions predicted to be highly frustrated were found to correlate with decreased GroEL dependence. Conversely, mutations at positions with low frustration were found to correlate with increased GroEL dependence. Further support for this finding was obtained by showing that folding of an enhanced green fluorescent protein variant designed computationally to have reduced frustration is indeed less GroEL-dependent. Our results indicate that changes in local frustration also affect partitioning in vivo between spontaneous and chaperonin-mediated folding. Hence, the design of minimally frustrated sequences can reduce chaperonin dependence and improve protein expression levels.

Overcoming an optimization plateau in the directed evolution of highly efficient nerve agent bioscavengers.

Improving an enzyme's initially low catalytic efficiency with a new target substrate by an order of magnitude or two may require only a few rounds of mutagenesis and screening or selection. However, subsequent rounds of optimization tend to yield decreasing degrees of improvement (diminishing returns) eventually leading to an optimization plateau. We aimed to optimize the catalytic efficiency of bacterial phosphotriesterase (PTE) toward V-type nerve agents. Previously, we improved the catalytic efficiency of wild-type PTE toward the nerve agent VX by 500-fold, to a catalytic efficiency (kcat/KM) of 5 × 106 M-1 min-1. However, effective in vivo detoxification demands an enzyme with a catalytic efficiency of >107 M-1 min-1. Here, following eight additional rounds of directed evolution and the computational design of a stabilized variant, we evolved PTE variants that detoxify VX with a kcat/KM ≥ 5 × 107 M-1 min-1 and Russian VX (RVX) with a kcat/KM ≥ 107 M-1 min-1. These final 10-fold improvements were the most time consuming and laborious, as most libraries yielded either minor or no improvements. Stabilizing the evolving enzyme, and avoiding tradeoffs in activity with different substrates, enabled us to obtain further improvements beyond the optimization plateau and evolve PTE variants that were overall improved by >5000-fold with VX and by >17 000-fold with RVX. The resulting variants also hydrolyze G-type nerve agents with high efficiency (GA, GB at kcat/KM > 5 × 107 M-1 min-1) and can thus serve as candidates for broad-spectrum nerve-agent prophylaxis and post-exposure therapy using low enzyme doses.

One-step design of a stable variant of the malaria invasion protein RH5 for use as a vaccine immunogen.

Many promising vaccine candidates from pathogenic viruses, bacteria, and parasites are unstable and cannot be produced cheaply for clinical use. For instance, Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) is essential for erythrocyte invasion, is highly conserved among field isolates, and elicits antibodies that neutralize in vitro and protect in an animal model, making it a leading malaria vaccine candidate. However, functional RH5 is only expressible in eukaryotic systems and exhibits moderate temperature tolerance, limiting its usefulness in hot and low-income countries where malaria prevails. Current approaches to immunogen stabilization involve iterative application of rational or semirational design, random mutagenesis, and biochemical characterization. Typically, each round of optimization yields minor improvement in stability, and multiple rounds are required. In contrast, we developed a one-step design strategy using phylogenetic analysis and Rosetta atomistic calculations to design PfRH5 variants with improved packing and surface polarity. To demonstrate the robustness of this approach, we tested three PfRH5 designs, all of which showed improved stability relative to wild type. The best, bearing 18 mutations relative to PfRH5, expressed in a folded form in bacteria at >1 mg of protein per L of culture, and had 10-15 °C higher thermal tolerance than wild type, while also retaining ligand binding and immunogenic properties indistinguishable from wild type, proving its value as an immunogen for a future generation of vaccines against the malaria blood stage. We envision that this efficient computational stability design methodology will also be used to enhance the biophysical properties of other recalcitrant vaccine candidates from emerging pathogens.

Automated Structure- and Sequence-Based Design of Proteins for High Bacterial Expression and Stability.

Upon heterologous overexpression, many proteins misfold or aggregate, thus resulting in low functional yields. Human acetylcholinesterase (hAChE), an enzyme mediating synaptic transmission, is a typical case of a human protein that necessitates mammalian systems to obtain functional expression. We developed a computational strategy and designed an AChE variant bearing 51 mutations that improved core packing, surface polarity, and backbone rigidity. This variant expressed at ∼2,000-fold higher levels in E. coli compared to wild-type hAChE and exhibited 20°C higher thermostability with no change in enzymatic properties or in the active-site configuration as determined by crystallography. To demonstrate broad utility, we similarly designed four other human and bacterial proteins. Testing at most three designs per protein, we obtained enhanced stability and/or higher yields of soluble and active protein in E. coli. Our algorithm requires only a 3D structure and several dozen sequences of naturally occurring homologs, and is available at http://pross.weizmann.ac.il.