Crucial roles of single residues in binding affinity, specificity, and promiscuity in the cellulosomal cohesin-dockerin interface.

Interactions between cohesin and dockerin modules play a crucial role in the assembly of multienzyme cellulosome complexes. Although intraspecies cohesin and dockerin modules bind in general with high affinity but indiscriminately, cross-species binding is rare. Here, we combined ELISA-based experiments with Rosetta-based computational design to evaluate the contribution of distinct residues at the Clostridium thermocellum cohesin-dockerin interface to binding affinity, specificity, and promiscuity. We found that single mutations can show distinct and significant effects on binding affinity and specificity. In particular, mutations at cohesin position Asn(37) show dramatic variability in their effect on dockerin binding affinity and specificity: the N37A mutant binds promiscuously both to cognate (C. thermocellum) as well as to non-cognate Clostridium cellulolyticum dockerin. N37L in turn switches binding specificity: compared with the wild-type C. thermocellum cohesin, this mutant shows significantly increased preference for C. cellulolyticum dockerin combined with strongly reduced binding to its cognate C. thermocellum dockerin. The observation that a single mutation can overcome the naturally observed specificity barrier provides insights into the evolutionary dynamics of this system that allows rapid modulation of binding specificity within a high affinity background.

Increased sequence conservation of domain repeats in prokaryotic proteins.

Protein domain repeats within a protein sequence have been observed throughout all domains of life. Our analysis shows a significantly higher degree of sequence identity between repeated domains in prokaryotes compared to eukaryotes. We discuss this difference in the light of aggregation prevention, contribution to functional divergence and binding-related functions. We then address the possible underlying features that create and conserve domain repeats. Our findings provide a starting point for the identification of the fundamental principles that underlie this basic difference between eukaryotic and prokaryotic protein evolution.

Polyclonal lymphoid expansion drives paraneoplastic autoimmunity in neuroblastoma.

Neuroblastoma is a lethal childhood solid tumor of developing peripheral nerves. Two percent of children with neuroblastoma develop opsoclonus myoclonus ataxia syndrome (OMAS), a paraneoplastic disease characterized by cerebellar and brainstem-directed autoimmunity but typically with outstanding cancer-related outcomes. We compared tumor transcriptomes and tumor-infiltrating T and B cell repertoires from 38 OMAS subjects with neuroblastoma to 26 non-OMAS-associated neuroblastomas. We found greater B and T cell infiltration in OMAS-associated tumors compared to controls and showed that both were polyclonal expansions. Tertiary lymphoid structures (TLSs) were enriched in OMAS-associated tumors. We identified significant enrichment of the major histocompatibility complex (MHC) class II allele HLA-DOB∗01:01 in OMAS patients. OMAS severity scores were associated with the expression of several candidate autoimmune genes. We propose a model in which polyclonal auto-reactive B lymphocytes act as antigen-presenting cells and drive TLS formation, thereby supporting both sustained polyclonal T cell-mediated anti-tumor immunity and paraneoplastic OMAS neuropathology.

Indirect ELISA-based approach for comparative measurement of high-affinity cohesin-dockerin interactions.

The interaction between the cohesin and dockerin modules serves to attach cellulolytic enzymes (carrying dockerins) to non-catalytic scaffoldin units (carrying multiple cohesins) in cellulosome, a multienzyme plant cell-wall degrading complex. This interaction is species-specific, for example, the enzyme-borne dockerin from Clostridium thermocellum bacteria binds to scaffoldin cohesins from the same bacteria but not to cohesins from Clostridium cellulolyticum and vice versa. We studied the role of interface residues, contributing either to affinity or specificity, by mutating these residues on the cohesin counterpart from C. thermocellum. The high affinity of the cognate interactions makes it difficult to evaluate the effect of these mutations by common methods used for measuring protein-protein interactions, especially when subtle discrimination between the mutants is needed. We described in this article an approach based on indirect enzyme-linked immunosorbent assay (ELISA) that is able to detect differences in binding between the various cohesin mutants, whereas surface plasmon resonance and standard ELISA failed to distinguish between high-affinity interactions. To be able to calculate changes in energy of binding (ΔΔG) and dissociation constants (K(d)) of mutants relative to wild type, a pre-equilibrium step was added to the standard indirect ELISA procedure. Thus, the cohesin-dockerin interaction under investigation occurs in solution rather than between soluble and immobilized proteins. Unbound dockerins are then detected through their interaction with immobilized cohesins. Because our method allows us to assess the effect of mutations on particularly tenacious protein-protein interactions much more accurately than do other prevalent methods used to measure binding affinity, we therefore suggest this approach as a method of choice for comparing relative binding in high-affinity interactions.

A hierarchy of selection pressures determines the organization of the T cell receptor repertoire.

We systematically examine the receptor repertoire in T cell subsets in young, adult, and LCMV-infected mice. Somatic recombination generates diversity, resulting in the limited overlap between nucleotide sequences of different repertoires even within the same individual. However, statistical features of the repertoire, quantified by the V gene and CDR3 k-mer frequency distributions, are highly conserved. A hierarchy of immunological processes drives the evolution of this structure. Intra-thymic divergence of CD4+ and CD8+ lineages imposes subtle but dominant differences observed across repertoires of all subpopulations in both young and adult mice. Differentiation from naive through memory to effector phenotype imposes an additional gradient of repertoire diversification, which is further influenced by age in a complex and lineage-dependent manner. The distinct repertoire of CD4+ regulatory T cells is more similar to naive cells in young mice and to effectors in adults. Finally, we describe divergent (naive and memory) and convergent (CD8+ effector) evolution of the repertoire following acute infection with LCMV. This study presents a quantitative framework that captures the structure of the repertoire in terms of its fundamental statistical properties and describes how this structure evolves as individual T cells differentiate, migrate and mature in response to antigen exposure.

Structure-based identification of catalytic residues.

The identification of catalytic residues is an essential step in functional characterization of enzymes. We present a purely structural approach to this problem, which is motivated by the difficulty of evolution-based methods to annotate structural genomics targets that have few or no homologs in the databases. Our approach combines a state-of-the-art support vector machine (SVM) classifier with novel structural features that augment structural clues by spatial averaging and Z scoring. Special attention is paid to the class imbalance problem that stems from the overwhelming number of non-catalytic residues in enzymes compared to catalytic residues. This problem is tackled by: (1) optimizing the classifier to maximize a performance criterion that considers both Type I and Type II errors in the classification of catalytic and non-catalytic residues; (2) under-sampling non-catalytic residues before SVM training; and (3) during SVM training, penalizing errors in learning catalytic residues more than errors in learning non-catalytic residues. Tested on four enzyme datasets, one specifically designed by us to mimic the structural genomics scenario and three previously evaluated datasets, our structure-based classifier is never inferior to similar structure-based classifiers and comparable to classifiers that use both structural and evolutionary features. In addition to the evaluation of the performance of catalytic residue identification, we also present detailed case studies on three proteins. This analysis suggests that many false positive predictions may correspond to binding sites and other functional residues. A web server that implements the method, our own-designed database, and the source code of the programs are publicly available at http://www.cs.bgu.ac.il/∼meshi/functionPrediction.

Myocardial infarction triggers cardioprotective antigen-specific T helper cell responses.

T cell autoreactivity is a hallmark of autoimmune diseases but can also benefit self-maintenance and foster tissue repair. Herein, we investigated whether heart-specific T cells exert salutary or detrimental effects in the context of myocardial infarction (MI), the leading cause of death worldwide. After screening more than 150 class-II-restricted epitopes, we found that myosin heavy chain alpha (MYHCA) was a dominant cardiac antigen triggering post-MI CD4+ T cell activation in mice. Transferred MYHCA614-629-specific CD4+ T (TCR-M) cells selectively accumulated in the myocardium and mediastinal lymph nodes (med-LN) of infarcted mice, acquired a Treg phenotype with a distinct pro-healing gene expression profile, and mediated cardioprotection. Myocardial Treg cells were also detected in autopsies from patients who suffered a MI. Noninvasive PET/CT imaging using a CXCR4 radioligand revealed enlarged med-LNs with increased cellularity in MI-patients. Notably, the med-LN alterations observed in MI patients correlated with the infarct size and cardiac function. Taken together, the results obtained in our study provide evidence showing that MI-context induces pro-healing T cell autoimmunity in mice and confirms the existence of an analogous heart/med-LN/T cell axis in MI patients.

The Genomic and Immune Landscapes of Lethal Metastatic Breast Cancer.

The detailed molecular characterization of lethal cancers is a prerequisite to understanding resistance to therapy and escape from cancer immunoediting. We performed extensive multi-platform profiling of multi-regional metastases in autopsies from 10 patients with therapy-resistant breast cancer. The integrated genomic and immune landscapes show that metastases propagate and evolve as communities of clones, reveal their predicted neo-antigen landscapes, and show that they can accumulate HLA loss of heterozygosity (LOH). The data further identify variable tumor microenvironments and reveal, through analyses of T cell receptor repertoires, that adaptive immune responses appear to co-evolve with the metastatic genomes. These findings reveal in fine detail the landscapes of lethal metastatic breast cancer.

Nuclear export of Ho endonuclease of yeast via Msn5.

Exportin-5, an evolutionarily conserved nuclear export factor of the beta-karyopherin family, exports phosphorylated proteins and small noncoding RNAs. Msn5, the yeast ortholog, exports primarily phosphorylated cargoes including Ho endonuclease and a number of transcription factors and regulatory proteins. The Msn5-mediated nuclear export of Ho is dependent on phosphorylation of Thr225 by kinases of the DNA damage response pathway. Although Msn5 has been the object of many studies, no NES sequence capable of binding the exportin and/or of leading to Msn5-dependent export of a heterologous protein has been identified. Here we report identification of a 13-residue Ho sequence that interacts with Msn5 in vitro and directs Msn5-dependent nuclear export of GFP in vivo. A single point mutation in this 13-mer Ho NES abrogates both interaction with Msn5 and nuclear export of Ho and of GFP. However, this mutation, or of T225A, both of which abrogate nuclear export of Ho, does not interfere with its interaction with Msn5 implying that the exportin makes multiple contacts with its cargo. This can explain the lack of a conserved NES in Msn5 cargoes. Our results identify essential criteria for Msn5-mediated nuclear export of Ho: phosphorylation on HoT225, and interaction with the 13-mer Ho NES sequence.

Combined Analysis of Antigen Presentation and T-cell Recognition Reveals Restricted Immune Responses in Melanoma.

The quest for tumor-associated antigens (TAA) and neoantigens is a major focus of cancer immunotherapy. Here, we combine a neoantigen prediction pipeline and human leukocyte antigen (HLA) peptidomics to identify TAAs and neoantigens in 16 tumors derived from seven patients with melanoma and characterize their interactions with their tumor-infiltrating lymphocytes (TIL). Our investigation of the antigenic and T-cell landscapes encompassing the TAA and neoantigen signatures, their immune reactivity, and their corresponding T-cell identities provides the first comprehensive analysis of cancer cell T-cell cosignatures, allowing us to discover remarkable antigenic and TIL similarities between metastases from the same patient. Furthermore, we reveal that two neoantigen-specific clonotypes killed 90% of autologous melanoma cells, both in vitro and in vivo, showing that a limited set of neoantigen-specific T cells may play a central role in melanoma tumor rejection. Our findings indicate that combining HLA peptidomics with neoantigen predictions allows robust identification of targetable neoantigens, which could successfully guide personalized cancer immunotherapies.Significance: As neoantigen targeting is becoming more established as a powerful therapeutic approach, investigating these molecules has taken center stage. Here, we show that a limited set of neoantigen-specific T cells mediates tumor rejection, suggesting that identifying just a few antigens and their corresponding T-cell clones could guide personalized immunotherapy. Cancer Discov; 8(11); 1366-75. ©2018 AACR. This article is highlighted in the In This Issue feature, p. 1333.

Measurements of relative binding of cohesin and dockerin mutants using an advanced ELISA technique for high-affinity interactions.

The cellulosome is a large bacterial extracellular multienzyme complex able to degrade crystalline cellulosic substrates. The complex contains catalytic and noncatalytic subunits, interconnected by high-affinity cohesin-dockerin interactions. In this chapter, we introduce an optimized method for comparative binding among different cohesins or cohesin mutants to the dockerin partner. This assay offers advantages over other methods (such as ELISA, cELIA, SPR, and ITC) for particularly high-affinity binding interactions. In this approach, the high-affinity interaction of interest occurs in the liquid phase during the equilibrated binding step, whereas the interaction with the immobilized phase is used only for detection of the unbound dockerins that remain in the solution phase. Once equilibrium conditions are reached, the change in free energy of binding (ΔΔG(binding)), as well as the affinity constant of mutants, can be estimated against the known affinity constant of the wild-type interaction. In light of the above, we propose this method as a preferred alternative for the relative quantification of high-affinity protein interactions.

The SV40 capsid is stabilized by a conserved pentapeptide hinge of the major capsid protein VP1.

The simian virus 40 (SV40) outer shell is composed of 72 pentamers of VP1. The core of the VP1 monomer is a beta-barrel with jelly-roll topology and extending N- and C-terminal arms. A pentapeptide hinge, KNPYP, tethers the C-arm to the VP1 beta-barrel core. The five C-arms that extend from each pentamer insert into the neighbouring pentamers, tying them together through different types of interactions. In the mature virion, this element adopts either of six conformations according to their location in the capsid. We found that the hinge is conserved among 16 members of the Polyomaviridae, attesting to its importance in capsid assembly and/or structure. We have used site-directed mutagenesis to gain an understanding into the structural requirements of this element: Y299 was changed to A, F, and T, and P300 to A and G. The mutants showed reduction in viability to varying degrees. Unexpectedly, assembly was reduced only to a small extent. However, the data showed that the mutants were highly unstable. The largest effect was observed for mutations of P300, indicating a role of the proline in the virion structure. P300G was more unstable than P300A, indicating a requirement for rigidity of the pentapeptide hinge. Y299T and Y299A were more defective in viability than Y299F, highlighting the importance of an aromatic ring at this position. Structural inspection showed that this aromatic ring contacts C-arms of neighbouring pentamers. Computational modelling predicted loss of stability of the Y mutants in concordance with the experimental results. This study provides insights into the structural details of the pentapeptide hinge that are responsible for capsid stability.

MESHI: a new library of Java classes for molecular modeling.

UNLABELLED: Adapting a modular and object-oriented approach in the design of molecular modeling packages may reduce the software development barrier between ideas and their programed applications. Towards this goal we developed MESHI, a new, strictly object-oriented, molecular modeling suite written in Java. MESHI provides a comprehensive library of extendable classes for all the essential components of molecular modeling: molecular and geometry elements, energy functions and optimization methods. AVAILABILITY: MESHI and its related documentation are freely available at http://www.cs.bgu.ac.il/~meshi; the MESHI API is available at http://www.cs.bgu.ac.il/~meshi/API CONTACT: keasar@cs.bgu.ac.il SUPPLEMENTARY INFORMATION: The Supplementary information includes (1) a detailed description of several key packages and classes, and (2) a brief presentation of results achieved by using the MESHI application--Beautify--in the CASP6 experiment.