Quaternary Architectures Research Articles

Simulations reveal unique roles for the FXR hinge in the FXR-RXR nuclear receptor heterodimer.

Nuclear receptors are multidomain transcription factors whose full-length quaternary architecture is poorly described and understood. Most nuclear receptors bind DNA as heterodimers or homodimers, which could encompass a variety of arrangements of the individual domains. Only a handful of experimental structures currently exist describing these architectures. Given that domain interactions and protein-DNA interactions within transcriptional complexes are tightly linked to function, understanding the arrangement of nuclear receptor domains on DNA is of utmost importance. Here, we employ modeling and molecular dynamics (MD) simulations to describe the structure of the full-length farnesoid X receptor (FXR) and retinoid X receptor alpha (RXR) heterodimer bound to DNA. Combining over 100 microseconds of atomistic MD simulations with enhanced sampling simulations, we characterize the dynamic behavior of eight FXR-RXR-DNA complexes, showing that these complexes support a range of quaternary architectures. Our simulations reveal critical roles for the hinge in the DNA-bound dimer in facilitating interdomain allostery, mediating DNA binding and driving the dynamic flexibility of the complex. These roles have been hard to appreciate previously due to experimental limitations in studying the flexible hinge. These studies provide a much-needed framework that will enable the field to obtain a complete understanding of nuclear receptor quaternary architectures.

Translational regulation by Hfq–Crc assemblies emerges from polymorphic ribonucleoprotein folding

The widely occurring bacterial RNA chaperone Hfq is a key factor in the post‐transcriptional control of hundreds of genes in Pseudomonas aeruginosa. How this broadly acting protein can contribute to the regulatory requirements of many different genes remains puzzling. Here, we describe cryo‐EM structures of higher order assemblies formed by Hfq and its partner protein Crc on control regions of different P. aeruginosa target mRNAs. Our results show that these assemblies have mRNA‐specific quaternary architectures resulting from the combination of multivalent protein–protein interfaces and recognition of patterns in the RNA sequence. The structural polymorphism of these ribonucleoprotein assemblies enables selective translational repression of many different target mRNAs. This system elucidates how highly complex regulatory pathways can evolve with a minimal economy of proteinogenic components in combination with RNA sequence and fold.

Structures of NPAS4-ARNT and NPAS4-ARNT2 heterodimers reveal new dimerization modalities in the bHLH-PAS transcription factor family

Neuronal PER-ARNT-SIM (PAS) domain protein 4 (NPAS4) is a protective transcriptional regulator whose dysfunction has been linked to a variety of neuropsychiatric and metabolic diseases. As a member of the basic helix-loop-helix PER-ARNT-SIM (bHLH-PAS) transcription factor family, NPAS4 is distinguished by an ability to form functional heterodimers with aryl hydrocarbon receptor nuclear translocator (ARNT) and ARNT2, both of which are also bHLH-PAS family members. Here, we describe the quaternary architectures of NPAS4-ARNT and NPAS4-ARNT2 heterodimers in complexes involving DNA response elements. Our crystallographic studies reveal a uniquely interconnected domain conformation for the NPAS4 protein itself, as well as its differentially configured heterodimeric arrangements with both ARNT and ARNT2. Notably, the PAS-A domains of ARNT and ARNT2 exhibit variable conformations within these two heterodimers. The ARNT PAS-A domain also forms a set of interfaces with the PAS-A and PAS-B domains of NPAS4, different from those previously noted in ARNT heterodimers formed with other class I bHLH-PAS family proteins. Our structural observations together with biochemical and cell-based interrogations of these NPAS4 heterodimers provide molecular glimpses of the NPAS4 protein architecture and extend the known repertoire of heterodimerization patterns within the bHLH-PAS family. The PAS-B domains of NPAS4, ARNT, and ARNT2 all contain ligand-accessible pockets with appropriate volumes required for small-molecule binding. Given NPAS4's linkage to human diseases, the direct visualization of these PAS domains and the further understanding of their relative positioning and interconnections within the NPAS4-ARNT and NPAS4-ARNT2 heterodimers may provide a road map for therapeutic discovery targeting these complexes.

Retinoic acid receptor structures: the journey from single domains to full-length complex.

The retinoic acid receptors (RARα, β, and γ) are multi-domain polypeptides that heterodimerize with retinoid X receptors (RXRα, β, and γ) to form functional transcription factors. Understanding the three-dimensional molecular organization of these nuclear receptors (NRs) began with RAR and RXR DNA-binding domains (DBDs), and were followed with studies on isolated ligand-binding domains (LBDs). The more complete picture emerged in 2017 with the multi-domain crystal structure of RXRα-RARβ on its response element with retinoic acid molecules and coactivator segments on both proteins. The analysis of that structure and its complementary studies have clarified the direct communication pathways within RXR-RAR polypeptides, through which DNA binding, protein-ligand, and protein-protein interactions are integrated for overall functional responses. Understanding the molecular connections in the RXR-RAR complex has benefited from direct observations of the multi-domain structures of RXRα-PPARγ, RXRα-LXRβ, HNF-4α homodimer, and androgen receptor homodimer, each bound to its response element. These comprehensive NR structures show unique quaternary architectures, yet all have DBD-DBD, LBD-LBD, and DBD-LBD domain-domain contacts within them. These convergence zones allow signals from discrete domains of their polypeptides to be propagated and integrated across their entire complex, shaping their overall responses in an allosteric fashion.

Fabrication of Hierarchical Quaternary Architectures of Metal-Organic Frameworks through Programmed Transformation.

A new method to construct hierarchical architectures has been developed by programmed transformation of metal-organic frameworks (MOFs). A MOF precursor was fabricated by reaction of Cu(OAC)2 and 2,5-dihydroxyterephthalic acid (H4DOBDC), which could perform transformation in pure methanol solvent and the sodium dodecyl benzene sulfonate (SDBS) solution of methanol, respectively. Interestingly, two kinds of immersion solutions could induce the transformation of the MOF precursor into MOF-74, which resulted in different morphologies: nanoneedles for the methanol and nanosheets for the SDBS. Herein, nanosheets-mesorods-microcuboid hierarchical quaternary architectures of MOF have been successfully achieved by sequential immersion of the precursor in two kinds of transformation solutions, which demonstrates well-defined hierarchy from the nanoscale to mesoscale to microscale. A unique hierarchical architecture could be recognized as quaternary structures, taking the MOF unit cell as the primary structure, the nanosheets as the secondary structure, the mesorods as the tertiary structure, and the microcuboid as the quaternary structure. Our study indicated the potential of programmed transformation between MOFs in the construction of hierarchical architectures, offering a new approach to sophisticated materials.

Symmetry breaking and structural polymorphism in a bacterial microcompartment shell protein for choline utilization.

Bacterial microcompartments are protein-based organelles that carry out specialized metabolic functions in diverse bacteria. Their outer shells are built from several thousand protein subunits. Some of the architectural principles of bacterial microcompartments have been articulated, with lateral packing of flat hexameric BMC proteins providing the basic foundation for assembly. Nonetheless, a complete understanding has been elusive, partly owing to polymorphic mechanisms of assembly exhibited by most microcompartment types. An earlier study of one homologous BMC shell protein subfamily, EutS/PduU, revealed a profoundly bent, rather than flat, hexameric structure. The possibility of a specialized architectural role was hypothesized, but artifactual effects of crystallization could not be ruled out. Here we report a series of crystal structures of an orthologous protein, CutR, from a glycyl-radical type choline-utilizing microcompartment from the bacterium Streptococcus intermedius. Depending on crystal form, expression construct, and minor mutations, a range of novel quaternary architectures was observed, including two spiral hexagonal assemblies. A new graphical approach helps illuminate the variations in BMC hexameric structure, with results substantiating the idea that the EutS/PduU/CutR subfamily of BMC proteins may endow microcompartment shells with flexible modes of assembly.

Plasmodium vivax and human hexokinases share similar active sites but display distinct quaternary architectures.

Malaria is a devastating disease caused by a protozoan parasite. It affects over 300 million individuals and results in over 400 000 deaths annually, most of whom are young children under the age of five. Hexokinase, the first enzyme in glucose metabolism, plays an important role in the infection process and represents a promising target for therapeutic intervention. Here, cryo-EM structures of two conformational states of Plasmodium vivax hexokinase (PvHK) are reported at resolutions of ∼3 Å. It is shown that unlike other known hexokinase structures, PvHK displays a unique tetrameric organization (∼220 kDa) that can exist in either open or closed quaternary conformational states. Despite the resemblance of the active site of PvHK to its mammalian counterparts, this tetrameric organization is distinct from that of human hexokinases, providing a foundation for the structure-guided design of parasite-selective antimalarial drugs.

Hierarchy in Metal-Organic Frameworks.

Sequence-defined nucleic acids and proteins with internal monomer sequences and arrangement are vital components in the living world, as a result of billions of years of molecular evolution. These natural hierarchical systems have inspired researchers to develop artificial hierarchical materials that can mimic similar functions such as replication, recognition, and information storage. In this Outlook, we describe the conceptual introduction of hierarchy into the design of metal–organic framework (MOF) materials. Starting with a history and background of hierarchical MOF synthesis and applications, we discuss further mesoscopic assembly strategies of MOF crystallites into hierarchical primary, secondary, tertiary, and quaternary architectures. This is followed by a highlight of the utilization of modular total synthesis for crafting MOFs with hierarchical compositions. The multiscale control over hierarchical MOF architecture formation can be rationally achieved by designing stepwise synthetic routes based on the knowledge from various fields including coordination chemistry, organic chemistry, reticular chemistry, and nanoscience. Altogether, this outlook is expected to shed light on these essential but embryonic materials and might offer inspiration for the development of the next generation of smart MOF materials with controllable heterogeneity and tailorable architectures.

Seed-mediated evolution of hierarchical metal-organic framework quaternary superstructures.

The idea of hierarchy, widely observed in natural and artificial worlds, has been extensively explored in chemistry and materials science. Similar to proteins which contain primary, secondary, tertiary and quaternary structures, varying levels of hierarchy in metal-organic framework (MOF) superstructures can also be achieved. In this work, we initiate a systematic study on the morphological evolution of hierarchical superstructures with the assistance of seeded growth and explore the assembly of multiple modular MOFs into superstructures with enhanced hierarchy and diversity. By utilizing MOF-74-III spherulite superstructures as seeds, multiple quaternary architectures were obtained depending on the lengths of organic linker precursors. The resulting superstructures with superior hierarchy represent a unique porous material which contains multiple modules with diverse morphologies. To the best of our knowledge, this is the first report that utilizes tertiary superstructures as seeds in MOF synthesis, which leads to unusual and diverse behaviors during secondary growth. This synthetic approach not only provides a facile method to establish hierarchy in porous materials, but also enables the fabrication of multiscale heterostructures through secondary growth on MOF seeds.

Evolutionary Morphing of Tryptophan Synthase: Functional Mechanisms for the Enzymatic Channeling of Indole

Tryptophan synthase (TrpS) is a heterotetrameric αββα enzyme that exhibits complex substrate channeling and allosteric mechanisms and is a model system in enzymology. In this work, we characterize proposed early and late evolutionary states of TrpS and show that they have distinct quaternary structures caused by insertions–deletions of sequence segments (indels) in the β-subunit. Remarkably, indole hydrophobic channels that connect α and β active sites have re-emerged in both TrpS types, yet they follow different paths through the β-subunit fold. Also, both TrpS geometries activate the α-subunit through the rearrangement of loops flanking the active site. Our results link evolutionary sequence changes in the enzyme subunits with channeling and allostery in the TrpS enzymes. The findings demonstrate that indels allow protein quaternary architectures to escape “minima” in the evolutionary landscape, thereby overcoming the conservational constraints imposed by existing functional interfaces and being free to morph into new mechanistic enzymes.

The quaternary architecture of RAR\u03b2\u2013RXR\u03b1 heterodimer facilitates domain\u2013domain signal transmission

Assessing the physical connections and allosteric communications in multi-domain nuclear receptor (NR) polypeptides has remained challenging, with few crystal structures available to show their overall structural organizations. Here we report the quaternary architecture of multi-domain retinoic acid receptor β–retinoic X receptor α (RARβ–RXRα) heterodimer bound to DNA, ligands and coactivator peptides, examined through crystallographic, hydrogen–deuterium exchange mass spectrometry, mutagenesis and functional studies. The RARβ ligand-binding domain (LBD) and DNA-binding domain (DBD) are physically connected to foster allosteric signal transmission between them. Direct comparisons among all the multi-domain NRs studied crystallographically to date show significant variations within their quaternary architectures, rather than a common architecture adhering to strict rules. RXR remains flexible and adaptive by maintaining loosely organized domains, while its heterodimerization partners use a surface patch on their LBDs to form domain-domain interactions with DBDs.

Understanding nuclear receptor form and function using structural biology

Nuclear receptors (NRs) are a major transcription factor family whose members selectively bind small-molecule lipophilic ligands and transduce those signals into specific changes in gene programs. For over two decades, structural biology efforts were focused exclusively on the individual ligand-binding domains (LBDs) or DNA-binding domains of NRs. These analyses revealed the basis for both ligand and DNA binding and also revealed receptor conformations representing both the activated and repressed states. Additionally, crystallographic studies explained how NR LBD surfaces recognize discrete portions of transcriptional coregulators. The many structural snapshots of LBDs have also guided the development of synthetic ligands with therapeutic potential. Yet, the exclusive structural focus on isolated NR domains has made it difficult to conceptualize how all the NR polypeptide segments are coordinated physically and functionally in the context of receptor quaternary architectures. Newly emerged crystal structures of the peroxisome proliferator-activated receptor-γ-retinoid X receptor α (PPARγ-RXRα) heterodimer and hepatocyte nuclear factor (HNF)-4α homodimer have recently revealed the higher order organizations of these receptor complexes on DNA, as well as the complexity and uniqueness of their domain-domain interfaces. These emerging structural advances promise to better explain how signals in one domain can be allosterically transmitted to distal receptor domains, also providing much better frameworks for guiding future drug discovery efforts.

Leonidas Zervas award lecture: Abiotic ligands for new quaternary architectures of peptides and proteins

This review is the result of a kind invitation to provide a report on the basis of the Leonidas Zervas award lecture at the 32nd European Peptide Society in Athens, Greece, September 2012. The lecture covered several topics including contributions toward new methods for the assembly of peptides and chemical modification of proteins. The present review will focus on another topic from the Zervas lecture, namely, our recent efforts at creating new, artificial architectures for the organization of the quaternary structure of proteins. Crucially, this is achieved with abiotic ligands and not protein surface engineering. This concept was demonstrated for the important biopharmaceutical peptide insulin, by using both abiotic metal ion binding ligands and perfluoroalkyl chains.

A novel method to map and compare protein–protein interactions in spherical viral capsids

Viral capsids are composed of multiple copies of one or a few chemically distinct capsid proteins and are mostly stabilized by inter subunit protein-protein interactions. There have been efforts to identify and analyze these protein-protein interactions, in terms of their extent and similarity, between the subunit interfaces related by quasi- and icosahedral symmetry. Here, we describe a new method to map quaternary interactions in spherical virus capsids onto polar angle space with respect to the icosahedral symmetry axes using azimuthal orthographic diagrams. This approach enables one to map the nonredundant interactions in a spherical virus capsid, irrespective of its size or triangulation number (T), onto the reference icosahedral asymmetric unit space. The resultant diagrams represent characteristic fingerprints of quaternary interactions of the respective capsids. Hence, they can be used as road maps of the protein-protein interactions to visualize the distribution and the density of the interactions. In addition, unlike the previous studies, the fingerprints of different capsids, when represented in a matrix form, can be compared with one another to quantitatively evaluate the similarity (S-score) in the subunit environments and the associated protein-protein interactions. The S-score selectively distinguishes the similarity, or lack of it, in the locations of the quaternary interactions as opposed to other well-known structural similarity metrics (e.g., RMSD, TM-score). Application of this method on a subset of T = 1 and T = 3 capsids suggests that S-score values range between 1 and 0.6 for capsids that belong to the same virus family/genus; 0.6-0.3 for capsids from different families with the same T-number and similar subunit fold; and <0.3 for comparisons of the dissimilar capsids that display different quaternary architectures (T-numbers). Finally, the sequence conserved interface residues within a virus family, whose spatial locations were also conserved have been hypothesized as the essential residues for self-assembly of the member virus capsids.

Molecular characterization, 3D modelling and expression analysis of sea bass ( Dicentrarchus labrax L.) interleukin-10

Interleukin-10 (IL-10) is a pleiotropic cytokine generally known for its relevance in the resolution of inflammation, but that also has immunostimulatory properties. Here is described the isolation and characterization of the sea bass IL-10 (sbIL-10) cDNA and gene. The sbIL-10 gene encodes a 187 amino acid protein and comprises a five exon-four intron structure as other known IL-10 genes. Important structural residues are maintained in the sbIL-10 protein, including the four cysteines responsible for the two intra-chain disulfide bridges reported for human IL-10. The 3D structure of sbIL-10 was predicted. This first homology model of a fish IL-10 reveals a high degree of compatibility between the dimeric quaternary architectures of sbIL-10 and its mammalian counterparts. The phylogenetic analysis clusters sbIL-10 with other IL-10s, apart from IL-10-related molecules. The involvement of IL-10 in sea bass immune responses was demonstrated by investigating the expression profiles of IL-1β and IL-10 in the head-kidney and spleen following intraperitoneal injection of UV-killed Photobacterium damselae ssp. piscicida. Furthermore, involvement of IL-10 in the resolution of inflammation is for the first time suggested in fish, due to the delayed maximal mRNA levels of sbIL-10 compared to those of the pro-inflammatory IL-1β.

Rebuilt AAA + motors reveal operating principles for ATP-fuelled machines

Hexameric ring-shaped ATPases of the AAA + (for ATPases associated with various cellular activities) superfamily power cellular processes in which macromolecular structures and complexes are dismantled or denatured, but the mechanisms used by these machine-like enzymes are poorly understood. By covalently linking active and inactive subunits of the ATPase ClpX to form hexamers, here we show that diverse geometric arrangements can support the enzymatic unfolding of protein substrates and translocation of the denatured polypeptide into the ClpP peptidase for degradation. These studies indicate that the ClpX power stroke is generated by ATP hydrolysis in a single subunit, rule out concerted and strict sequential ATP hydrolysis models, and provide evidence for a probabilistic sequence of nucleotide hydrolysis. This mechanism would allow any ClpX subunit in contact with a translocating polypeptide to hydrolyse ATP to drive substrate spooling into ClpP, and would prevent stalling if one subunit failed to bind or hydrolyse ATP. Energy-dependent machines with highly diverse quaternary architectures and molecular functions could operate by similar asymmetric mechanisms.