Asymmetric mutations in the tetrameric R67 dihydrofolate reductase reveal high tolerance to active-site substitutions

Type II R67 dihydrofolate reductase (DHFR) is a bacterial plasmid-encoded enzyme that is intrinsically resistant to the widely-administered antibiotic trimethoprim. R67 DHFR is genetically and structurally unrelated to E. coli chromosomal DHFR and has an unusual architecture, in that four identical protomers form a single symmetrical active site tunnel that allows only one substrate binding/catalytic event at any given time. As a result, substitution of an active-site residue has as many as four distinct consequences on catalysis, constituting an atypical model of enzyme evolution. Although we previously demonstrated that no single residue of the native active site is indispensable for function, library selection here revealed a strong bias toward maintenance of two native protomers per mutated tetramer. A variety of such “half-native” tetramers were shown to procure native-like catalytic activity, with similar K_M values but k_cat values 5- to 33-fold lower, illustrating a high tolerance for active-site substitutions. The selected variants showed a reduced thermal stability (T_m ∼12°C lower), which appears to result from looser association of the protomers, but generally showed a marked increase in resilience to heat denaturation, recovering activity to a significantly greater extent than the variant with no active-site substitutions. Our results suggest that the presence of two native protomers in the R67 DHFR tetramer is sufficient to provide native-like catalytic rate and thus ensure cellular proliferation.     

Differentiation of the binding of two ligands to a tetrameric protein with a single symmetric active site by 19F NMR

R67 dihydrofolate reductase (R67 DHFR) is a plasmid‐encoded enzyme that confers resistance to the antibacterial drug trimethoprim. R67 DHFR is a tetramer with a single active site that is unusual as both cofactor and substrate are recognized by symmetry‐related residues. Such promiscuity has limited our previous efforts to differentiate ligand binding by NMR. To address this problem, we incorporated fluorine at positions 4, 5, 6, or 7 of the indole rings of tryptophans 38 and 45 and characterized the spectra to determine which probe was optimal for studying ligand binding. Two resonances were observed for all apo proteins. Unexpectedly, the W45 resonance appeared broad, and truncation of the disordered N‐termini resulted in the appearance of one sharp W45 resonance. These results are consistent with interaction of the N‐terminus with W45. Binding of the cofactor broadened W38 for all fluorine probes, whereas substrate, dihydrofolate, binding resulted in the appearance of three new resonances for 4‐ and 5‐fluoroindole labeled protein and severe line broadening for 6‐ and 7‐fluoroindole R67 DHFR. W45 became slightly broader upon ligand binding. With only two peaks in the ¹⁹F NMR spectra, our data were able to differentiate cofactor and substrate binding to the single, symmetric active site of R67 DHFR and yield binding affinities.     

The importance of bottlenecks in protein networks: correlation with gene essentiality and expression dynamics

It has been a long-standing goal in systems biology to find relations between the topological properties and functional features of protein networks. However, most of the focus in network studies has been on highly connected proteins (“hubs”). As a complementary notion, it is possible to define bottlenecks as proteins with a high betweenness centrality (i.e., network nodes that have many “shortest paths” going through them, analogous to major bridges and tunnels on a highway map). Bottlenecks are, in fact, key connector proteins with surprising functional and dynamic properties. In particular, they are more likely to be essential proteins. In fact, in regulatory and other directed networks, betweenness (i.e., “bottleneck-ness”) is a much more significant indicator of essentiality than degree (i.e., “hub-ness”). Furthermore, bottlenecks correspond to the dynamic components of the interaction network—they are significantly less well coexpressed with their neighbors than nonbottlenecks, implying that expression dynamics is wired into the network topology.     

Glycan-dependent and -independent Interactions Contribute to Cellular Substrate Recruitment by Calreticulin

Calreticulin is an endoplasmic reticulum chaperone with specificity for monoglucosylated glycoproteins. Calreticulin also inhibits precipitation of nonglycosylated proteins and thus contains generic protein-binding sites, but their location and contributions to substrate folding are unknown. We show that calreticulin binds glycosylated and nonglycosylated proteins with similar affinities but distinct interaction kinetics. Although both interactions involve the glycan-binding site or its vicinity, the arm-like proline-rich (P-) domain of calreticulin contributes to binding non/deglycosylated proteins. Correspondingly, ensemble FRET spectroscopy measurements indicate that glycosylated and nonglycosylated proteins induce “open” and “closed” P-domain conformations, respectively. The co-chaperone ERp57 influences substrate-binding kinetics and induces a closed P-domain conformation. Together with analysis of the interactions of calreticulin with cellular proteins, these findings indicate that the recruitment of monoglucosylated proteins to calreticulin is kinetically driven, whereas the P-domain and co-chaperone contribute to stable substrate binding. Substrate sequestration in the cleft between the glycan-binding site and P-domain is a likely mechanism for calreticulin-assisted protein folding.     

Exploring the Morphospace of Communication Efficiency in Complex Networks

Graph theoretical analysis has played a key role in characterizing global features of the topology of complex networks, describing diverse systems such as protein interactions, food webs, social relations and brain connectivity. How system elements communicate with each other depends not only on the structure of the network, but also on the nature of the system''s dynamics which are constrained by the amount of knowledge and resources available for communication processes. Complementing widely used measures that capture efficiency under the assumption that communication preferentially follows shortest paths across the network (“routing”), we define analytic measures directed at characterizing network communication when signals flow in a random walk process (“diffusion”). The two dimensions of routing and diffusion efficiency define a morphospace for complex networks, with different network topologies characterized by different combinations of efficiency measures and thus occupying different regions of this space. We explore the relation of network topologies and efficiency measures by examining canonical network models, by evolving networks using a multi-objective optimization strategy, and by investigating real-world network data sets. Within the efficiency morphospace, specific aspects of network topology that differentially favor efficient communication for routing and diffusion processes are identified. Charting regions of the morphospace that are occupied by canonical, evolved or real networks allows inferences about the limits of communication efficiency imposed by connectivity and dynamics, as well as the underlying selection pressures that have shaped network topology.     

Mapping and Quantitation of the Interaction between the Recombination Activating Gene Proteins RAG1 and RAG2

The RAG endonuclease consists of RAG1, which contains the active site for DNA cleavage, and RAG2, an accessory factor whose interaction with RAG1 is critical for catalytic function. How RAG2 activates RAG1 is not understood. Here, we used biolayer interferometry and pulldown assays to identify regions of RAG1 necessary for interaction with RAG2 and to measure the RAG1-RAG2 binding affinity (K_D ∼0.4 μm) (where RAG1 and RAG2 are recombination activating genes 1 or 2). Using the Hermes transposase as a guide, we constructed a 36-kDa “mini” RAG1 capable of interacting robustly with RAG2. Mini-RAG1 consists primarily of the catalytic center and the residues N-terminal to it, but it lacks a zinc finger region in RAG1 previously implicated in binding RAG2. The ability of Mini-RAG1 to interact with RAG2 depends on a predicted α-helix (amino acids 997–1008) near the RAG1 C terminus and a region of RAG1 from amino acids 479 to 559. Two adjacent acidic amino acids in this region (Asp-546 and Glu-547) are important for both the RAG1-RAG2 interaction and recombination activity, with Asp-546 of particular importance. Structural modeling of Mini-RAG1 suggests that Asp-546/Glu-547 lie near the predicted 997-1008 α-helix and components of the active site, raising the possibility that RAG2 binding alters the structure of the RAG1 active site. Quantitative Western blotting allowed us to estimate that mouse thymocytes contain on average ∼1,800 monomers of RAG1 and ∼15,000 molecules of RAG2, implying that nuclear concentrations of RAG1 and RAG2 are below the K_D value for their interaction, which could help limit off-target RAG activity.     

De novo protein fold families expand the designable ligand binding site space

A major challenge in designing proteins de novo to bind user-defined ligands with high affinity is finding backbones structures into which a new binding site geometry can be engineered with high precision. Recent advances in methods to generate protein fold families de novo have expanded the space of accessible protein structures, but it is not clear to what extend de novo proteins with diverse geometries also expand the space of designable ligand binding functions. We constructed a library of 25,806 high-quality ligand binding sites and developed a fast protocol to place (“match”) these binding sites into both naturally occurring and de novo protein families with two fold topologies: Rossman and NTF2. Each matching step involves engineering new binding site residues into each protein “scaffold”, which is distinct from the problem of comparing already existing binding pockets. 5,896 and 7,475 binding sites could be matched to the Rossmann and NTF2 fold families, respectively. De novo designed Rossman and NTF2 protein families can support 1,791 and 678 binding sites that cannot be matched to naturally existing structures with the same topologies, respectively. While the number of protein residues in ligand binding sites is the major determinant of matching success, ligand size and primary sequence separation of binding site residues also play important roles. The number of matched binding sites are power law functions of the number of members in a fold family. Our results suggest that de novo sampling of geometric variations on diverse fold topologies can significantly expand the space of designable ligand binding sites for a wealth of possible new protein functions.     

Structure-Based Network Analysis of Activation Mechanisms in the ErbB Family of Receptor Tyrosine Kinases: The Regulatory Spine Residues Are Global Mediators of Structural Stability and Allosteric Interactions

The ErbB protein tyrosine kinases are among the most important cell signaling families and mutation-induced modulation of their activity is associated with diverse functions in biological networks and human disease. We have combined molecular dynamics simulations of the ErbB kinases with the protein structure network modeling to characterize the reorganization of the residue interaction networks during conformational equilibrium changes in the normal and oncogenic forms. Structural stability and network analyses have identified local communities integrated around high centrality sites that correspond to the regulatory spine residues. This analysis has provided a quantitative insight to the mechanism of mutation-induced “superacceptor” activity in oncogenic EGFR dimers. We have found that kinase activation may be determined by allosteric interactions between modules of structurally stable residues that synchronize the dynamics in the nucleotide binding site and the αC-helix with the collective motions of the integrating αF-helix and the substrate binding site. The results of this study have pointed to a central role of the conserved His-Arg-Asp (HRD) motif in the catalytic loop and the Asp-Phe-Gly (DFG) motif as key mediators of structural stability and allosteric communications in the ErbB kinases. We have determined that residues that are indispensable for kinase regulation and catalysis often corresponded to the high centrality nodes within the protein structure network and could be distinguished by their unique network signatures. The optimal communication pathways are also controlled by these nodes and may ensure efficient allosteric signaling in the functional kinase state. Structure-based network analysis has quantified subtle effects of ATP binding on conformational dynamics and stability of the EGFR structures. Consistent with the NMR studies, we have found that nucleotide-induced modulation of the residue interaction networks is not limited to the ATP site, and may enhance allosteric cooperativity with the substrate binding region by increasing communication capabilities of mediating residues.     

Effect of cofactor binding and loop conformation on side chain methyl dynamics in dihydrofolate reductase

Dihydrofolate reductase (DHFR) has several flexible active site loops that facilitate ligand binding and catalysis. Previous studies of backbone dynamics in several complexes of DHFR indicate that the time scale and amplitude of motion depend on the conformation of the active site loops. In this study, information on dynamics is extended to methyl-containing side chains. To understand the role of side chain dynamics in ligand binding and loop conformation, methyl deuterium relaxation rates of Escherichia coli DHFR in binary folate and ternary folate:NADP+ complexes have been measured, together with chi(1) rotamer populations for threonine, isoleucine, and valine residues, determined from measurements of 3J(CgammaCO) and 3J(CgammaN) coupling constants. The results indicate that, in addition to backbone motional restriction in the adenosine-binding site, side chain flexibility in the active site and the surrounding active site loops is diminished upon binding NADP+. Resonances for several methyls in the active site and the surrounding active site loops were severely broadened in the folate:NADP+ ternary complex, suggesting the presence of motion on the chemical shift time scale. The side chains of Ile14 and Ile94, which pack against the nicotinamide and pterin rings of the cofactor and substrate, respectively, exhibit rotamer disorder in the ternary folate:NADP+ complex. Conformational fluctuations of these side chains may play a role in transition state stabilization; the observed line broadening for Ile14 suggests motions on a microsecond/millisecond time scale.     

Allosteric Communication Occurs via Networks of Tertiary and Quaternary Motions in Proteins

Allosteric proteins bind an effector molecule at one site resulting in a functional change at a second site. We hypothesize that allosteric communication in proteins relies upon networks of quaternary (collective, rigid-body) and tertiary (residue–residue contact) motions. We argue that cyclic topology of these networks is necessary for allosteric communication. An automated algorithm identifies rigid bodies from the displacement between the inactive and the active structures and constructs “quaternary networks” from these rigid bodies and the substrate and effector ligands. We then integrate quaternary networks with a coarse-grained representation of contact rearrangements to form “global communication networks” (GCNs). The GCN reveals allosteric communication among all substrate and effector sites in 15 of 18 multidomain and multimeric proteins, while tertiary and quaternary networks exhibit such communication in only 4 and 3 of these proteins, respectively. Furthermore, in 7 of the 15 proteins connected by the GCN, 50% or more of the substrate-effector paths via the GCN are “interdependent” paths that do not exist via either the tertiary or the quaternary network. Substrate-effector “pathways” typically are not linear but rather consist of polycyclic networks of rigid bodies and clusters of rearranging residue contacts. These results argue for broad applicability of allosteric communication based on structural changes and demonstrate the utility of the GCN. Global communication networks may inform a variety of experiments on allosteric proteins as well as the design of allostery into non-allosteric proteins.     

A Pareto-Optimal Refinement Method for Protein Design Scaffolds

Computational design of protein function involves a search for amino acids with the lowest energy subject to a set of constraints specifying function. In many cases a set of natural protein backbone structures, or “scaffolds”, are searched to find regions where functional sites (an enzyme active site, ligand binding pocket, protein – protein interaction region, etc.) can be placed, and the identities of the surrounding amino acids are optimized to satisfy functional constraints. Input native protein structures almost invariably have regions that score very poorly with the design force field, and any design based on these unmodified structures may result in mutations away from the native sequence solely as a result of the energetic strain. Because the input structure is already a stable protein, it is desirable to keep the total number of mutations to a minimum and to avoid mutations resulting from poorly-scoring input structures. Here we describe a protocol using cycles of minimization with combined backbone/sidechain restraints that is Pareto-optimal with respect to RMSD to the native structure and energetic strain reduction. The protocol should be broadly useful in the preparation of scaffold libraries for functional site design.     

Electrostatically Accelerated Encounter and Folding for Facile Recognition of Intrinsically Disordered Proteins

Achieving facile specific recognition is essential for intrinsically disordered proteins (IDPs) that are involved in cellular signaling and regulation. Consideration of the physical time scales of protein folding and diffusion-limited protein-protein encounter has suggested that the frequent requirement of protein folding for specific IDP recognition could lead to kinetic bottlenecks. How IDPs overcome such potential kinetic bottlenecks to viably function in signaling and regulation in general is poorly understood. Our recent computational and experimental study of cell-cycle regulator p27 (Ganguly et al., J. Mol. Biol. (2012)) demonstrated that long-range electrostatic forces exerted on enriched charges of IDPs could accelerate protein-protein encounter via “electrostatic steering” and at the same time promote “folding-competent” encounter topologies to enhance the efficiency of IDP folding upon encounter. Here, we further investigated the coupled binding and folding mechanisms and the roles of electrostatic forces in the formation of three IDP complexes with more complex folded topologies. The surface electrostatic potentials of these complexes lack prominent features like those observed for the p27/Cdk2/cyclin A complex to directly suggest the ability of electrostatic forces to facilitate folding upon encounter. Nonetheless, similar electrostatically accelerated encounter and folding mechanisms were consistently predicted for all three complexes using topology-based coarse-grained simulations. Together with our previous analysis of charge distributions in known IDP complexes, our results support a prevalent role of electrostatic interactions in promoting efficient coupled binding and folding for facile specific recognition. These results also suggest that there is likely a co-evolution of IDP folded topology, charge characteristics, and coupled binding and folding mechanisms, driven at least partially by the need to achieve fast association kinetics for cellular signaling and regulation.     

Role of the AP2 β-Appendage Hub in Recruiting Partners for Clathrin-Coated Vesicle Assembly

Adaptor protein complex 2 α and β-appendage domains act as hubs for the assembly of accessory protein networks involved in clathrin-coated vesicle formation. We identify a large repertoire of β-appendage interactors by mass spectrometry. These interact with two distinct ligand interaction sites on the β-appendage (the “top” and “side” sites) that bind motifs distinct from those previously identified on the α-appendage. We solved the structure of the β-appendage with a peptide from the accessory protein Eps15 bound to the side site and with a peptide from the accessory cargo adaptor β-arrestin bound to the top site. We show that accessory proteins can bind simultaneously to multiple appendages, allowing these to cooperate in enhancing ligand avidities that appear to be irreversible in vitro. We now propose that clathrin, which interacts with the β-appendage, achieves ligand displacement in vivo by self-polymerisation as the coated pit matures. This changes the interaction environment from liquid-phase, affinity-driven interactions, to interactions driven by solid-phase stability (“matricity”). Accessory proteins that interact solely with the appendages are thereby displaced to areas of the coated pit where clathrin has not yet polymerised. However, proteins such as β-arrestin (non-visual arrestin) and autosomal recessive hypercholesterolemia protein, which have direct clathrin interactions, will remain in the coated pits with their interacting receptors.     

Explicating the Face Perception Network with White Matter Connectivity

A network of multiple brain regions is recruited in face perception. Our understanding of the functional properties of this network can be facilitated by explicating the structural white matter connections that exist between its functional nodes. We accomplished this using functional MRI (fMRI) in combination with fiber tractography on high angular resolution diffusion weighted imaging data. We identified the three nodes of the core face network: the “occipital face area” (OFA), the “fusiform face area” (mid-fusiform gyrus or mFus), and the superior temporal sulcus (STS). Additionally, a region of the anterior temporal lobe (aIT), implicated as being important for face perception was identified. Our data suggest that we can further divide the OFA into multiple anatomically distinct clusters – a partitioning consistent with several recent neuroimaging results. More generally, structural white matter connectivity within this network revealed: 1) Connectivity between aIT and mFus, and between aIT and occipital regions, consistent with studies implicating this posterior to anterior pathway as critical to normal face processing; 2) Strong connectivity between mFus and each of the occipital face-selective regions, suggesting that these three areas may subserve different functional roles; 3) Almost no connectivity between STS and mFus, or between STS and the other face-selective regions. Overall, our findings suggest a re-evaluation of the “core” face network with respect to what functional areas are or are not included in this network.     

Intrinsic connectivity reveals functionally distinct cortico-hippocampal networks in the human brain

Episodic memory depends on interactions between the hippocampus and interconnected neocortical regions. Here, using data-driven analyses of resting-state functional magnetic resonance imaging (fMRI) data, we identified the networks that interact with the hippocampus—the default mode network (DMN) and a “medial temporal network” (MTN) that included regions in the medial temporal lobe (MTL) and precuneus. We observed that the MTN plays a critical role in connecting the visual network to the DMN and hippocampus. The DMN could be further divided into 3 subnetworks: a “posterior medial” (PM) subnetwork comprised of posterior cingulate and lateral parietal cortices; an “anterior temporal” (AT) subnetwork comprised of regions in the temporopolar and dorsomedial prefrontal cortex; and a “medial prefrontal” (MP) subnetwork comprised of regions primarily in the medial prefrontal cortex (mPFC). These networks vary in their functional connectivity (FC) along the hippocampal long axis and represent different kinds of information during memory-guided decision-making. Finally, a Neurosynth meta-analysis of fMRI studies suggests new hypotheses regarding the functions of the MTN and DMN subnetworks, providing a framework to guide future research on the neural architecture of episodic memory.

Episodic memory depends on interactions between the hippocampus and interconnected neocortical regions. This study uses network analyses of intrinsic brain networks at rest to identify and characterize brain networks that interact with the hippocampus and have distinct functions during memory-guided decision making.     

Structural Insights into the Catalytic Mechanism of Synechocystis Magnesium Protoporphyrin IX O-Methyltransferase (ChlM)

Magnesium protoporphyrin IX O-methyltransferase (ChlM) catalyzes transfer of the methyl group from S-adenosylmethionine to the carboxyl group of the C13 propionate side chain of magnesium protoporphyrin IX. This reaction is the second committed step in chlorophyll biosynthesis from protoporphyrin IX. Here we report the crystal structures of ChlM from the cyanobacterium Synechocystis sp. PCC 6803 in complex with S-adenosylmethionine and S-adenosylhomocysteine at resolutions of 1.6 and 1.7 Å, respectively. The structures illustrate the molecular basis for cofactor and substrate binding and suggest that conformational changes of the two “arm” regions may modulate binding and release of substrates/products to and from the active site. Tyr-28 and His-139 were identified to play essential roles for methyl transfer reaction but are not indispensable for cofactor/substrate binding. Based on these structural and functional findings, a catalytic model is proposed.     

Polymorphisms in the Human Inhibitory Signal-regulatory Protein α Do Not Affect Binding to Its Ligand CD47

CD47 is a widely distributed membrane protein that interacts with signal-regulatory protein α (SIRPα), an inhibitory receptor on myeloid cells that gives a “don''t-eat-me” signal. Manipulation of the interaction is of considerable interest in the immunotherapy of cancer and in xenotransplantation. The amino-terminal ligand binding domain of SIRPα is highly polymorphic in contrast to the single Ig-like domain of CD47. There is confusion as to whether the polymorphisms will affect ligand binding, but this is an important point for this interaction and other paired receptors being considered as targets for therapy. We show by x-ray crystallography that one human SIRPα allele differing in 13 amino acid residues has a very similar binding site and that several different alleles all bind CD47 with similar affinity as expected because the residues are mostly surface-exposed and distant from the binding site. A peptide from the binding site of CD47 has been reported to mimic the CD47 interaction with SIRPα, but we could find no binding. We discuss the possible pitfalls in determining the affinity of weak interactions and also speculate on how SIRPα polymorphisms may have been selected by pathogens and how this may also be true in other paired receptors such as the KIRs.     

Not all scale-free networks are born equal: the role of the seed graph in PPI network evolution

The (asymptotic) degree distributions of the best-known “scale-free” network models are all similar and are independent of the seed graph used; hence, it has been tempting to assume that networks generated by these models are generally similar. In this paper, we observe that several key topological features of such networks depend heavily on the specific model and the seed graph used. Furthermore, we show that starting with the “right” seed graph (typically a dense subgraph of the protein–protein interaction network analyzed), the duplication model captures many topological features of publicly available protein–protein interaction networks very well.     

Plasticity of Human Protein Disulfide Isomerase: EVIDENCE FOR MOBILITY AROUND THE X-LINKER REGION AND ITS FUNCTIONAL SIGNIFICANCE*

Protein disulfide isomerase (PDI), which consists of multiple domains arranged as abb′xa′c, is a key enzyme responsible for oxidative folding in the endoplasmic reticulum. In this work we focus on the conformational plasticity of this enzyme. Proteolysis of native human PDI (hPDI) by several proteases consistently targets sites in the C-terminal half of the molecule (x-linker and a′ domain) leaving large fragments in which the N terminus is intact. Fluorescence studies on the W111F/W390F mutant of full-length PDI show that its fluorescence is dominated by Trp-347 in the x-linker which acts as an intrinsic reporter and indicates that this linker can move between “capped” and “uncapped” conformations in which it either occupies or exposes the major ligand binding site on the b′ domain of hPDI. Studies with a range of constructs and mutants using intrinsic fluorescence, collision quenching, and extrinsic probe fluorescence (1-anilino-8-naphthalene sulfonate) show that the presence of the a′ domain in full-length hPDI moderates the ability of the x-linker to generate the capped conformation (compared with shorter fragments) but does not abolish it. Hence, unlike yeast PDI, the major conformational plasticity of full-length hPDI concerns the mobility of the a′ domain “arm” relative to the bb′ “trunk” mediated by the x-linker. The chaperone and enzymatic activities of these constructs and mutants are consistent with the interpretation that the reversible interaction of the x-linker with the ligand binding site mediates access of protein substrates to this site.     

A structural intermediate pre-organizes the add adenine riboswitch for ligand recognition

Riboswitches are RNA sequences that regulate gene expression by undergoing structural changes upon the specific binding of cellular metabolites. Crystal structures of purine-sensing riboswitches have revealed an intricate network of interactions surrounding the ligand in the bound complex. The mechanistic details about how the aptamer folding pathway is involved in the formation of the metabolite binding site have been previously shown to be highly important for the riboswitch regulatory activity. Here, a combination of single-molecule FRET and SHAPE assays have been used to characterize the folding pathway of the adenine riboswitch from Vibrio vulnificus. Experimental evidences suggest a folding process characterized by the presence of a structural intermediate involved in ligand recognition. This intermediate state acts as an open conformation to ensure ligand accessibility to the aptamer and folds into a structure nearly identical to the ligand-bound complex through a series of structural changes. This study demonstrates that the add riboswitch relies on the folding of a structural intermediate that pre-organizes the aptamer global structure and the ligand binding site to allow efficient metabolite sensing and riboswitch genetic regulation.