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We describe the design, kinetic properties, and structures of engineered subtilisin proteases that degrade the active form of RAS by cleaving a conserved sequence in switch 2. RAS is a signaling protein that, when mutated, drives a third of human cancers. To generate high specificity for the RAS target sequence, the active site was modified to be dependent on a cofactor (imidazole or nitrite) and protease sub-sites were engineered to create a linkage between substrate and cofactor binding. Selective proteolysis of active RAS arises from a 2-step process wherein sub-site interactions promote productive binding of the cofactor, enabling cleavage. Proteases engineered in this way specifically cleave active RAS in vitro, deplete the level of RAS in a bacterial reporter system, and also degrade RAS in human cell culture. Although these proteases target active RAS, the underlying design principles are fundamental and will be adaptable to many target proteins., Chen et al. describe a rational design of subtilisin mutants that degrade active RAS by cleaving a conserved sequence in switch 2. They further modified the active site to be dependent on a cofactor to generate high target specificity. Proteases engineered to cleave this region degraded RAS in vitro and in cells with a promise of adaptability for other target proteins too.

As monoclonal antibodies have become a vital resource in medicine, knowledge of their complex molecular structures has increased in importance. Thousands of antibody components (Fab and Fc fragments) are described in the Protein Data Bank. Whole antibodies have been imaged by electron microscopy methods and in a few cases, crystallized. The central hinge lacks a unique stable conformation and its dynamic properties are important to antibody function. Monte Carlo and molecular dynamics simulations and small-angle scattering methods have been used to analyze the wide range of configurations that are accessible to antibodies in solution. In order to support the development of antibody-based medicines, the National Institute of Standards and Technology (NIST) has released an extensively characterized IgG1κ monoclonal antibody (mAb), called the NISTmAb Reference Material 8671. To facilitate modeling of whole antibodies we now report the construction of an all-atom 3-D model of the NISTmAb.

Adoptive cell therapy (ACT) with tumor-specific T cells can mediate cancer regression. The main target of tumor-specific T cells are neoantigens arising from mutations in self-proteins. Although the majority of cancer neoantigens are unique to each patient, and therefore not broadly useful for ACT, some are shared. We studied oligoclonal T-cell receptors (TCRs) that recognize a shared neoepitope arising from a driver mutation in the p53 oncogene (p53R175H) presented by HLA-A2. Here we report structures of wild-type and mutant p53–HLA-A2 ligands, as well as structures of three tumor-specific TCRs bound to p53R175H–HLA-A2. These structures reveal how a driver mutation in p53 rendered a self-peptide visible to T cells. The TCRs employ structurally distinct strategies that are highly focused on the mutation to discriminate between mutant and wild-type p53. The TCR–p53R175H–HLA-A2 complexes provide a framework for designing TCRs to improve potency for ACT without sacrificing specificity., Developing broadly applicable neoantigen-directed adoptive cell therapies (ACTs) is challenging because each cancer patient has an unique neoantigen repertoire. Here, the authors present the crystal structures of tumor-specific T cell receptors (TCRs) that recognize a shared neoepitope arising from the R175H driver mutation in the p53 oncogene (p53R175H) alone and bound to p53R175H–HLA-A2, which are of interest for the structure-guided design of TCRs to improve T cell potency for ACT.

The human chaperonin complex is a ~ 1 MDa nanomachine composed of two octameric rings formed from eight similar but non-identical subunits called CCT. Here, we are elucidating the mechanism of a heritable CCT5 subunit mutation that causes profound neuropathy in humans. In previous work, we introduced an equivalent mutation in an archaeal chaperonin that assembles into two octameric rings like in humans but in which all subunits are identical. We reported that the hexadecamer formed by the mutant subunit is unstable with impaired chaperoning functions. This study quantifies the loss of structural stability in the hexadecamer due to the pathogenic mutation, using differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC). The disassembly of the wild type complex, which is tightly coupled with subunit denaturation, was decoupled by the mutation without affecting the stability of individual subunits. Our results verify the effectiveness of the homo-hexadecameric archaeal chaperonin as a proxy to assess the impact of subtle defects in heterologous systems with mutations in a single subunit., Graphical abstract fx1, Highlights • A crippling hereditary neuropathy was addressed at the molecular level. • The archaeal/CCT5 model represents a promising testbed for subtle defects. • The homomeric archaeal model amplifies the effect of the mutation. • The mutation decouples assembly without destabilizing individual subunits.

Antibody-drug conjugates (ADCs) are a class of biotherapeutic drugs designed as targeted therapies for the treatment of cancer. Among the challenges in generating an effective ADC is the choice of an effective conjugation site on the IgG. One common method to prepare site-specific ADCs is to engineer solvent-accessible cysteine residues into antibodies. Here, we used X-ray diffraction and hydrogen-deuterium exchange mass spectroscopy to analyze the structure and dynamics of such a construct where a cysteine has been inserted after Ser 239 (Fc-239i) in the antibody heavy chain sequence. The crystal structure of this Fc-C239i variant at 0.23 nm resolution shows that the inserted cysteine structurally replaces Ser 239 and that this causes a domino-like backward shift of the local polypeptide, pushing Pro 238 out into the hinge. Proline is unable to substitute conformationally for the wild-type glycine at this position, providing a structural reason for the previously observed abolition of both Fc&gamma, R binding and antibody-dependent cellular cytotoxicity. Energy estimates for the both the Fc&gamma, R interface (7 kcal/mol) and for the differential conformation of proline (20 kcal/mol) are consistent with the observed disruption of Fc&gamma, R binding, providing a quantifiable case where strain at a single residue appears to disrupt a key biological function. Conversely, the structure of Fc-C239i is relatively unchanged at the intersection of the CH2 and CH3 domains, the site known to be involved in binding of the neonatal Fc receptor (FcRn), and an alignment of the Fc-C239i structure with an Fc structure in a ternary Fc:FcRn:HSA (human serum albumin) complex implies that these favorable contacts would be maintained. Hydrogen deuterium exchange mass spectroscopy (HDX-MS) data further suggest a significant increase in conformational mobility for the Fc-C239i protein relative to Fc that is evident even far from the insertion site but still largely confined to the CH2 domain. Together, the findings provide a detailed structural and dynamic basis for previously observed changes in ADC functional binding to Fc&gamma, R, which may guide further development of ADC designs.

Both conformational and colloidal stability of therapeutic proteins must be closely monitored and thoroughly characterized to assess the long-term viability of drug products. We characterized the IgG1 NISTmAb reference material in its histidine formulation buffer and report our findings on the higher order structure and interactions of NISTmAb under a range of conditions. In this paper we present the analysis of experimental small-angle scattering data with atomistic molecular simulations to characterize the monodisperse dilute solution of NISTmAb. In part II we describe the characterization of the NISTmAb at high protein concentration (Castellanos et al. 2018). The NISTmAb was found to be a flexible protein with a radius of gyration of 49.0 ± 1.2 A in histidine formulation buffer using a variety of neutron and X-ray scattering measurements. Scattering data were then modeled using molecular simulation. After building and validating a starting NISTmAb structure from the Fc and Fab crystallographic coordinates, molecular dynamics and torsion-angle Monte Carlo simulations were performed to explore the configuration space sampled in the NISTmAb and obtain ensembles of structures with atomistic detail that are consistent with the experimental data. Our results indicate that the small-angle scattering profiles of the NISTmAb can be modeled using ensembles of flexible structures that explore a wide configuration space. The NISTmAb is flexible in solution with no single preferred orientation of Fc and Fab domains, but with some regions of configuration space that are more consistent with measured scattering profiles. Analysis of inter-domain atomistic contacts indicated that all ensembles contained configurations where residues between domains are ≤ 4 A, although few contacts were observed for variable and C H 3 regions.

PlyC, a bacteriophage-encoded endolysin, lyses Streptococcus pyogenes (Spy) on contact. Here, we demonstrate that PlyC is a potent agent for controlling intracellular Spy that often underlies refractory infections. We show that the PlyC holoenzyme, mediated by its PlyCB subunit, crosses epithelial cell membranes and clears intracellular Spy in a dose-dependent manner. Quantitative studies using model membranes establish that PlyCB interacts strongly with phosphatidylserine (PS), whereas its interaction with other lipids is weak, suggesting specificity for PS as its cellular receptor. Neutron reflection further substantiates that PlyC penetrates bilayers above a PS threshold concentration. Crystallography and docking studies identify key residues that mediate PlyCB–PS interactions, which are validated by site-directed mutagenesis. This is the first report that a native endolysin can traverse epithelial membranes, thus substantiating the potential of PlyC as an antimicrobial for Spy in the extracellular and intracellular milieu and as a scaffold for engineering other functionalities. DOI: http://dx.doi.org/10.7554/eLife.13152.001, eLife digest Streptococcus pyogenes is the bacterium that causes throat infections and other serious infections in humans. Antibiotics such as penicillin are used to treat active infections, but so-called “strep throat infections” often return after treatment. This is because S. pyogenes can enter the cells that line the throat and hide from the antibiotics, which cannot enter the throat cells. Endolysins are enzymes produced by viruses that attack bacteria, and these enzymes target and destroy the bacterial cell wall. A previous study revealed that an endolysin known as PlyC could destroy S. pyogenes bacteria on contact. PlyC and other endolysins have the potential to act as alternatives to common antibiotics, but before these enzymes can be developed as therapeutics, it is important to understand how they interact with human host cells. Like antibiotics, the PlyC endolysin was not expected to enter throat cells. However, Shen, Barros et al. have now discovered that not only can PlyC enter throat cells, it can essentially chase down and kill S. pyogenes that are hiding inside. Other similar enzymes could not act in this way, and further studies confirmed that PlyC could move around inside a throat cell without causing it damage. Shen, Barros et al. also determined that PlyC has a pocket on its surface that binds with a specific component of the throat cell membrane, a molecule called phosphatidylserine. This interaction – which is a bit like a lock and key – grants PlyC access into the cell. While it is clear that PlyC eventually kills S. pyogenes hiding inside throat cells, future experiments will aim to determine how PlyC moves around once inside an infected throat cell. Together, an understanding of how an endolysin enters cells and destroys hiding S. pyogenes will contribute to the development of endolysins with broader activity, which can be used as alternatives to common antibiotics. DOI: http://dx.doi.org/10.7554/eLife.13152.002

Development of an ontology for the description of crystallization experiments and results is proposed., When crystallization screening is conducted many outcomes are observed but typically the only trial recorded in the literature is the condition that yielded the crystal(s) used for subsequent diffraction studies. The initial hit that was optimized and the results of all the other trials are lost. These missing results contain information that would be useful for an improved general understanding of crystallization. This paper provides a report of a crystallization data exchange (XDX) workshop organized by several international large-scale crystallization screening laboratories to discuss how this information may be captured and utilized. A group that administers a significant fraction of the world’s crystallization screening results was convened, together with chemical and structural data informaticians and computational scientists who specialize in creating and analysing large disparate data sets. The development of a crystallization ontology for the crystallization community was proposed. This paper (by the attendees of the workshop) provides the thoughts and rationale leading to this conclusion. This is brought to the attention of the wider audience of crystallographers so that they are aware of these early efforts and can contribute to the process going forward.