Rational Reprogramming of Fungal Polyketide First Ring Cyclization

Resorcylic acid lactones (RAL) and dihydroxyphenylacetic acid lactones (DAL) represent important pharmacophores with heat shock response and immune system modulatory activities. The biosynthesis of these fungal polyketides involves a pair of collaborating iterative polyketide synthases (iPKSs): a highly reducing iPKS (hrPKS) whose product is further elaborated by a nonreducing iPKS (nrPKS) to yield a 1,3-benzenediol moiety bridged by a macrolactone. Biosynthesis of unreduced polyketides requires the sequestration and programmed cyclization of highly reactive poly-β-ketoacyl intermediates to channel these uncommitted, pluripotent substrates towards defined subsets of the polyketide structural space. Catalyzed by product template (PT) domains of the fungal nrPKSs and discrete aromatase/cyclase enzymes in bacteria, regiospecific first-ring aldol cyclizations result in characteristically different polyketide folding modes. However, a few fungal polyketides, including the DAL dehydrocurvularin, derive from a folding event that is analogous to the bacterial folding mode. The structural basis of such a drastic difference in the way a PT domain acts has not been investigated until now. We report here that the fungal versus the bacterial folding mode difference is portable upon creating hybrid enzymes, and structurally characterize the resulting unnatural products. Using structure-guided active site engineering, we unravel structural contributions to regiospecific aldol condensations, and show that reshaping the cyclization chamber of a PT domain by only three selected point mutations is sufficient to reprogram the dehydrocurvularin nrPKS to produce polyketides with a fungal fold. Such rational control of first ring cyclizations will facilitate efforts towards the engineered biosynthesis of novel chemical diversity from natural unreduced polyketides

Communication breakdown : dissecting the COM interfaces between the subunits of nonribosomal peptide synthetases

Nonribosomal peptides are a structurally diverse and bioactive class of natural products constructed by multidomain enzymatic assembly lines known as nonribosomal peptide synthetases (NRPSs). While the core catalytic domains and even entire protein subunits of NRPSs have been structurally elucidated, little biophysical work has been reported on the docking domains that promote interactions—and thus transfer of biosynthetic intermediates—between subunits. In the present study, we closely examine the COM domains that mediate COMmunication between donor epimerization (E) and acceptor condensation (C) domains found at the termini of NRPS subunits. Through a combination of X-ray crystallography, circular dichroism spectroscopy, solution- and solid-state NMR spectroscopy, and molecular dynamics (MD) simulations, we provide direct evidence for an intrinsically disordered donor COM region that folds into a dynamic helical motif upon binding to a suitable acceptor. Furthermore, our NMR titration and carbene footprinting experiments illuminate the residues involved at the COM interaction interface, and our MD simulations demonstrate folding consistent with experimental data. Although our results lend credence to the previously proposed helix-hand mode of interaction, they also underscore the importance of viewing COM interfaces as dynamic ensembles rather than single rigid structures and suggest that engineering experiments should account for the interactions which transiently guide folding in addition to those which stabilize the final complex. Through activity assays and affinity measurements, we further substantiate the role of the donor COM region in binding the acceptor C domain and implicate this short motif as readily transposable for noncognate domain crosstalk. Finally, our bioinformatics analyses show that COM domains are widespread in natural product pathways and function at interfaces beyond the canonical type described above, setting a high priority for thorough characterization of these docking domains. Our findings lay the groundwork for future attempts to rationally engineer NRPS domain–domain interactions with the ultimate goal of generating bioactive molecules

CRIB effector disorder: exquisite function from chaos.

The CRIB (Cdc42/Rac interactive binding) family of small G-protein effectors contain significant regions with intrinsic disorder. The G-protein-binding regions are contained within these intrinsically disordered regions. Most CRIB proteins also contain stretches of basic residues associated with their G-protein-binding regions. The basic region (BR) and G-protein-binding region together allow the CRIB effectors to bind to their cognate G-protein via a dock- and coalesce-binding mechanism. The BRs of these proteins take on multiple roles: steering G-protein binding, interacting with elements of the membrane and regulating intramolecular regulatory interactions. The ability of these regions of the CRIBs to undergo multivalent interactions and mediate charge neutralizations equips them with all the properties required to drive liquid-liquid phase separation and therefore to initiate and drive signalosome formation. It is only recently that the structural plasticity in these proteins is being appreciated as the driving force for these vital cellular processes.MRC CASE studentship MR/K017101/

Disordered domains in chromatin-binding proteins.

Chromatin comprises proteins, DNA and RNA, and its function is to condense and package the genome in a way that allows the necessary transactions such as transcription, replication and repair to occur in a highly organised and regulated manner. The packaging of chromatin is often thought of in a hierarchical fashion starting from the most basic unit of DNA packaging, the nucleosome, to the condensation of nucleosomal 'beads on a string' by linker histones to form the 30-nm fibre and eventually large chromatin domains. However, a picture of a more heterogeneous, dynamic and liquid-like assembly is emerging, in which intrinsically disordered proteins (IDPs) and proteins containing intrinsically disordered regions (IDRs) play a central role. Disorder features at all levels of chromatin organisation, from the histone tails, which are sites of extensive post-translational modification (PTM) that change the fate of the underlying genomic information, right through to transcription hubs, and the recently elucidated roles of IDPs and IDRs in the condensation of large regions of the genome through liquid-liquid phase separation

Single-molecule experiments in biological physics: methods and applications

I review single-molecule experiments (SME) in biological physics. Recent technological developments have provided the tools to design and build scientific instruments of high enough sensitivity and precision to manipulate and visualize individual molecules and measure microscopic forces. Using SME it is possible to: manipulate molecules one at a time and measure distributions describing molecular properties; characterize the kinetics of biomolecular reactions and; detect molecular intermediates. SME provide the additional information about thermodynamics and kinetics of biomolecular processes. This complements information obtained in traditional bulk assays. In SME it is also possible to measure small energies and detect large Brownian deviations in biomolecular reactions, thereby offering new methods and systems to scrutinize the basic foundations of statistical mechanics. This review is written at a very introductory level emphasizing the importance of SME to scientists interested in knowing the common playground of ideas and the interdisciplinary topics accessible by these techniques. The review discusses SME from an experimental perspective, first exposing the most common experimental methodologies and later presenting various molecular systems where such techniques have been applied. I briefly discuss experimental techniques such as atomic-force microscopy (AFM), laser optical tweezers (LOT), magnetic tweezers (MT), biomembrane force probe (BFP) and single-molecule fluorescence (SMF). I then present several applications of SME to the study of nucleic acids (DNA, RNA and DNA condensation), proteins (protein-protein interactions, protein folding and molecular motors). Finally, I discuss applications of SME to the study of the nonequilibrium thermodynamics of small systems and the experimental verification of fluctuation theorems. I conclude with a discussion of open questions and future perspectives.Comment: Latex, 60 pages, 12 figures, Topical Review for J. Phys. C (Cond. Matt

Dissecting and reprogramming the folding and assembly of tandem-repeat proteins.

Studying protein folding and protein design in globular proteins presents significant challenges because of the two related features, topological complexity and co-operativity. In contrast, tandem-repeat proteins have regular and modular structures composed of linearly arrayed motifs. This means that the biophysics of even giant repeat proteins is highly amenable to dissection and to rational design. Here we discuss what has been learnt about the folding mechanisms of tandem-repeat proteins. The defining features that have emerged are: (i) accessibility of multiple distinct routes between denatured and native states, both at equilibrium and under kinetic conditions; (ii) different routes are favoured for folding compared with unfolding; (iii) unfolding energy barriers are broad, reflecting stepwise unravelling of an array repeat by repeat; (iv) highly co-operative unfolding at equilibrium and the potential for exceptionally high thermodynamic stabilities by introducing consensus residues; (v) under force, helical-repeat structures are very weak with non-co-operative unfolding leading to elasticity and buffering effects. This level of understanding should enable us to create repeat proteins with made-to-measure folding mechanisms, in which one can dial into the sequence the order of repeat folding, number of pathways taken, step size (co-operativity) and fine-structure of the kinetic energy barriers.We acknowledge funding from the Medical Research Council of the UK (grant G1002329) and the Leverhulme Trust. AP is funded by a BBSRC Doctoral Training Program studentship. LSI acknowledges support of a Fellowship from the Medical Research Foundation.This is the accepted manuscript. The final version is available at http://www.biochemsoctrans.org/content/43/5/881

Structural insights into the basis and evolution of interactions in multi-subunit protein assemblies. tryptophan synthase and titin FNIII-repeats

Cellular processes benefit from evolutionary shaping when optimized protein-protein interactions result in enhanced functionality. In fact, most cellular proteins are tightly embedded into biological networks that function following a modularity principle. Modularity, whether based on components as parts of stable protein complexes or as dynamic units that interact only transiently (as in signalling and metabolic cascades), facilitates the combinatorial generation of complexity in protein networks through the re-wiring of modules in addition to the diversification of individual proteins – thereby increasing the “evolvability” of the system. The mechanisms that drive the emergence and evolution of molecular recognition in protein networks remain unclear. It is difficult to justify such evolution on the basis of organismic advantage, since the latter might only be noticeable once full pathways and cascades have evolved. It is then likely that the evolution of protein-protein interactions is in the first instance driven by a molecular principle of local advantage to the protein system itself - for example, molecular stability. Unfortunately, it is difficult to gain insights into the evolution of protein-protein interactions since the pathways of evolutionary shaping normally let intermediates of evolution disappear. Subsequently, conclusions are more usually drawn from the comparison of proteins between different species and by mutagenesis probing. In the current study, we aim at gaining an insight into the evolutionary shaping of proteins surfaces for hetero-complex formation by studying two systems at an early stage of development: Tryptophan Synthase B2b (TrpB2b) from S. solfataricus and the modular interfaces of the poly-FNIII tandems in the muscle filament titin. In the case of TrpB2b, the evolution of inter-subunit communication is addressed in addition. Both structures have been elucidated using X-ray crystallography and a comparative analysis of their surfaces has been carried out. The architectural elements subjected to evolutionary pressure have been identified and conclusions on their relation to function and evolution have been drawn

Synthetic Supramolecular Systems in Life-like Materials and Protocell Models

One of the biggest challenges in modern chemistry is the preparation of synthetic materials with life-like behavior for the assembly of artificial cells. In recent years, numerous artificial systems that mimic cellular components and functions have been developed. Supramolecular chemistry plays a key role in such cell mimics given that non-covalent interactions control the shape and function of many biomolecules, such as DNA base pairing, protein structure, ligand-receptor binding, and lipid membrane packing. However, the complexity of living cells constitutes a major challenge for their bottom-up assembly from pure synthetic materials. Inspired by the building blocks of nature, a wide range of supramolecular systems have been developed to reproduce cellular functions such as cell-cell communication, signaling cascades, and dynamic cytoskeleton assemblies. This review surveys a selection of key advances in synthetic derivatives of biomolecules with supramolecular organization and life-like behavior by addressing their non-covalent foundation and integration as increasingly complex protocell modelsThis work was partially supported by the Spanish Agencia Estatal de Investigación (AEI; SAF2017-89890-R), Xunta de Galicia (ED431C 2017/25, 2016-AD031, and ED431G/09), ISCIII (RD16/0008/003), and the European Commission (EC; European Regional Development Fund). I.I. thanks the EC and AEI for MSCA-IF (2018-843332) and JdC (FJCI-2017-31795) fellowships, respectively. J.M. received a Ramón y Cajal grant (RYC-2013-13784), an ERC-Stg grant (DYNAP-677786), and a Young Investigator Grant from the Human Frontier Science Program (RGY0066/2017)S