High avidity drives the interaction between the streptococcal C1 phage endolysin, PlyC, with the cell surface carbohydrates of Group A Streptococcus.

Endolysin enzymes from bacteriophage cause bacterial lysis by degrading the peptidoglycan cell wall. The streptococcal C1 phage endolysin PlyC, is the most potent endolysin described to date and can rapidly lyse group A, C, and E streptococci. PlyC is known to bind the Group A streptococcal cell wall, but the specific molecular target or the binding site within PlyC remain uncharacterized. Here we report for the first time, that the polyrhamnose backbone of the Group A streptococcal cell wall is the binding target of PlyC. We have also characterized the putative rhamnose binding groove of PlyC and found four key residues that were critical to either the folding or the cell wall binding action of PlyC. Based on our results, we suggest that the interaction between PlyC and the cell wall may not be a high-affinity interaction as previously proposed, but rather a high avidity one, allowing for PlyC's remarkable lytic activity. Resistance to our current antibiotics is reaching crisis levels and there is an urgent need to develop the antibacterial agents with new modes of action. A detailed understanding of this potent endolysin may facilitate future developments of PlyC as a tool against the rise of antibiotic resistance.

Impact of HLA-DR Antigen Binding Cleft Rigidity on T Cell Recognition.

The interaction between T cell receptor (TCR) and peptide (p)-Human Leukocyte Antigen (HLA) complexes is the critical first step in determining T cell responses. X-ray crystallographic studies of pHLA in TCR-bound and free states provide a structural perspective that can help understand T cell activation. These structures represent a static "snapshot", yet the nature of pHLAs and their interactions with TCRs are highly dynamic. This has been demonstrated for HLA class I molecules with in silico techniques showing that some interactions, thought to stabilise pHLA-I, are only transient and prone to high flexibility. Here, we investigated the dynamics of HLA class II molecules by focusing on three allomorphs (HLA-DR1, -DR11 and -DR15) that are able to present the same epitope and activate CD4+ T cells. A single TCR (F24) has been shown to recognise all three HLA-DR molecules, albeit with different affinities. Using molecular dynamics and crystallographic ensemble refinement, we investigate the molecular basis of these different affinities and uncover hidden roles for HLA polymorphic residues. These polymorphisms were responsible for the widening of the antigen binding cleft and disruption of pHLA-TCR interactions, underpinning the hierarchy of F24 TCR binding affinity, and ultimately T cell activation. We expanded this approach to all available pHLA-DR structures and discovered that all HLA-DR molecules were inherently rigid. Together with in vitro protein stability and peptide affinity measurements, our results suggest that HLA-DR1 possesses inherently high protein stability, and low HLA-DM susceptibility.

Intrinsic limitations in mainstream methods of identifying network motifs in biology.

BACKGROUND: Network motifs are connectivity structures that occur with significantly higher frequency than chance, and are thought to play important roles in complex biological networks, for example in gene regulation, interactomes, and metabolomes. Network motifs may also become pivotal in the rational design and engineering of complex biological systems underpinning the field of synthetic biology. Distinguishing true motifs from arbitrary substructures, however, remains a challenge. RESULTS: Here we demonstrate both theoretically and empirically that implicit assumptions present in mainstream methods for motif identification do not necessarily hold, with the ramification that motif studies using these mainstream methods are less able to effectively differentiate between spurious results and events of true statistical significance than is often presented. We show that these difficulties cannot be overcome without revising the methods of statistical analysis used to identify motifs. CONCLUSIONS: Present-day methods for the discovery of network motifs, and, indeed, even the methods for defining what they are, are critically reliant on a set of incorrect assumptions, casting a doubt on the scientific validity of motif-driven discoveries. The implications of these findings are therefore far-reaching across diverse areas of biology.

The Role of Conformational Dynamics in Abacavir-Induced Hypersensitivity Syndrome.

Abacavir is an antiretroviral drug used to reduce human immunodeficiency virus (HIV) replication and decrease the risk of developing acquired immune deficiency syndrome (AIDS). However, its therapeutic value is diminished by the fact that it is associated with drug hypersensitivity reactions in up to 8% of treated patients. This hypersensitivity is strongly associated with patients carrying human leukocyte antigen (HLA)-B*57:01, but not patients carrying closely related alleles. Abacavir's specificity to HLA-B*57:01 is attributed to its binding site within the peptide-binding cleft and subsequent influence of the repertoire of peptides that can bind HLA-B*57:01. To further our understanding of abacavir-induced hypersensitivity we used molecular dynamics (MD) to analyze the dynamics of three different peptides bound to HLA-B*57:01 in the presence and absence of abacavir or abacavir analogues. We found that abacavir and associated peptides bind to HLA-B*57:01 in a highly diverse range of conformations that are not apparent from static crystallographic snapshots, but observed no difference in either the conformations, nor degree of flexibility when compared to abacavir-unbound systems. Our results support hypersensitivity models in which abacavir-binding alters the conformational ensemble of neopeptides, so as to favour exposed peptide surfaces that are no longer recognized as self by circulating CD8+ T cells, and are conducive to TCR binding. Our findings highlight the need to also consider the role of dynamics in understanding drug-induced hypersensitivities at the molecular and mechanistic level. This additional insight can help inform the chemical modification of abacavir to prevent hypersensitivity reactions in HLA-B*57:01+ HIV patients whilst retaining potent antiretroviral activity.

Reactive centre loop dynamics and serpin specificity.

Serine proteinase inhibitors (serpins), typically fold to a metastable native state and undergo a major conformational change in order to inhibit target proteases. However, conformational lability of the native serpin fold renders them susceptible to misfolding and aggregation, and underlies misfolding diseases such as α1-antitrypsin deficiency. Serpin specificity towards its protease target is dictated by its flexible and solvent exposed reactive centre loop (RCL), which forms the initial interaction with the target protease during inhibition. Previous studies have attempted to alter the specificity by mutating the RCL to that of a target serpin, but the rules governing specificity are not understood well enough yet to enable specificity to be engineered at will. In this paper, we use conserpin, a synthetic, thermostable serpin, as a model protein with which to investigate the determinants of serpin specificity by engineering its RCL. Replacing the RCL sequence with that from α1-antitrypsin fails to restore specificity against trypsin or human neutrophil elastase. Structural determination of the RCL-engineered conserpin and molecular dynamics simulations indicate that, although the RCL sequence may partially dictate specificity, local electrostatics and RCL dynamics may dictate the rate of insertion during protease inhibition, and thus whether it behaves as an inhibitor or a substrate. Engineering serpin specificity is therefore substantially more complex than solely manipulating the RCL sequence, and will require a more thorough understanding of how conformational dynamics achieves the delicate balance between stability, folding and function required by the exquisite serpin mechanism of action.

Previously Hidden Dynamics at the TCR-Peptide-MHC Interface Revealed.

A structural characterization of the interaction between αß TCRs and cognate peptide-MHC (pMHC) is central to understanding adaptive T cell-mediated immunity. X-ray crystallography, although the source of much structural data, traditionally provides only a static snapshot of the protein. Given the emerging evidence for the important role of conformational dynamics in protein function, we interrogated 309 crystallographic structures of pMHC complexes using ensemble refinement, a technique that can extract dynamic information from the x-ray data. Focusing on a subset of human pMHC class I systems, we found that in many cases, ensemble methods were able to uncover previously hidden evidence of significant conformational plasticity, thereby revealing additional information that can build upon and significantly enhance functional interpretations that are based on a single static structure. Notable examples include the interpretation of differences in the disease association of HLA subtypes, the relationship between peptide prominence and TCR recognition, the role of conformational flexibility in vaccine design, and the discrimination between induced fit and conformational selection models of TCR binding. We show that the currently widespread practice of analyzing pMHC interactions via the study of a single crystallographic structure does not make use of pertinent and easily accessible information from x-ray data concerning alternative protein conformations. This new analysis therefore not only highlights the capacity for ensemble methods to significantly enrich the interpretation of decades of structural data but also provides previously missing information concerning the dynamics of existing characterized TCR-pMHC interactions.

Modelling of Thyroid Peroxidase Reveals Insights into Its Enzyme Function and Autoantigenicity.

Thyroid peroxidase (TPO) catalyses the biosynthesis of thyroid hormones and is a major autoantigen in Hashimoto's disease--the most common organ-specific autoimmune disease. Epitope mapping studies have shown that the autoimmune response to TPO is directed mainly at two surface regions on the molecule: immunodominant regions A and B (IDR-A, and IDR-B). TPO has been a major target for structural studies for over 20 years; however, to date, the structure of TPO remains to be determined. We have used a molecular modelling approach to investigate plausible modes of TPO structure and dimer organisation. Sequence features of the C-terminus are consistent with a coiled-coil dimerization motif that most likely anchors the TPO dimer in the apical membrane of thyroid follicular cells. Two contrasting models of TPO were produced, differing in the orientation and exposure of their active sites relative to the membrane. Both models are equally plausible based upon the known enzymatic function of TPO. The "trans" model places IDR-B on the membrane-facing side of the myeloperoxidase (MPO)-like domain, potentially hindering access of autoantibodies, necessitating considerable conformational change, and perhaps even dissociation of the dimer into monomers. IDR-A spans MPO- and CCP-like domains and is relatively fragmented compared to IDR-B, therefore most likely requiring domain rearrangements in order to coalesce into one compact epitope. Less epitope fragmentation and higher solvent accessibility of the "cis" model favours it slightly over the "trans" model. Here, IDR-B clusters towards the surface of the MPO-like domain facing the thyroid follicular lumen preventing steric hindrance of autoantibodies. However, conformational rearrangements may still be necessary to allow full engagement with autoantibodies, with IDR-B on both models being close to the dimer interface. Taken together, the modelling highlights the need to consider the oligomeric state of TPO, its conformational properties, and its proximity to the membrane, when interpreting epitope-mapping data.