Dimerization and Heme Binding Are Conserved in Amphibian and Starfish Homologues of the microRNA Processing Protein DGCR8

Human DiGeorge Critical Region 8 (DGCR8) is an essential microRNA (miRNA) processing factor that is activated via direct interaction with Fe(III) heme. In order for DGCR8 to bind heme, it must dimerize using a dimerization domain embedded within its heme-binding domain (HBD). We previously reported a crystal structure of the dimerization domain from human DGCR8, which demonstrated how dimerization results in the formation of a surface important for association with heme. Here, in an attempt to crystallize the HBD, we search for DGCR8 homologues and show that DGCR8 from Patiria miniata (bat star) also binds heme. The extinction coefficients (ε) of DGCR8-heme complexes are determined; these values are useful for biochemical analyses and allow us to estimate the heme occupancy of DGCR8 proteins. Additionally, we present the crystal structure of the Xenopus laevis dimerization domain. The structure is very similar to that of human DGCR8. Our results indicate that dimerization and heme binding are evolutionarily conserved properties of DGCR8 homologues not only in vertebrates, but also in at least some invertebrates

Protonation state of glutamate 73 regulates the formation of a specific dimeric association of mVDAC1.

The voltage-dependent anion channel (VDAC) is the most abundant protein in the outer mitochondrial membrane and constitutes the primary pathway for the exchange of ions and metabolites between the cytosol and the mitochondria. There is accumulating evidence supporting VDAC's role in mitochondrial metabolic regulation and apoptosis, where VDAC oligomerization has been implicated with these processes. Herein, we report a specific pH-dependent dimerization of murine VDAC1 (mVDAC1) identified by double electron-electron resonance and native mass spectrometry. Intermolecular distances on four singly spin-labeled mVDAC1 mutants were used to generate a model of the low-pH dimer, establishing the presence of residue E73 at the interface. This dimer arrangement is different from any oligomeric state previously described, and it forms as a steep function of pH with an apparent pKa of 7.4. Moreover, the monomer-dimer equilibrium affinity constant was determined using native MS, revealing a nearly eightfold enhancement in dimerization affinity at low pH. Mutation of E73 to either alanine or glutamine severely reduces oligomerization, demonstrating the role of protonated E73 in enhancing dimer formation. Based on these results, and the known importance of E73 in VDAC physiology, VDAC dimerization likely plays a significant role in mitochondrial metabolic regulation and apoptosis in response to cytosolic acidification during cellular stress

Origin of Complexity in Hemoglobin Evolution

Most proteins associate into multimeric complexes with specific architectures, which often have functional properties such as cooperative ligand binding or allosteric regulation. No detailed knowledge is available about how any multimer and its functions arose during evolution. Here we use ancestral protein reconstruction and biophysical assays to elucidate the origins of vertebrate hemoglobin, a heterotetramer of paralogous α- and β-subunits that mediates respiratory oxygen transport and exchange by cooperatively binding oxygen with moderate affinity. We show that modern hemoglobin evolved from an ancient monomer and characterize the historical “missing link” through which the modern tetramer evolved—a noncooperative homodimer with high oxygen affinity that existed before the gene duplication that generated distinct α- and β-subunits. Reintroducing just two post-duplication historical substitutions into the ancestral protein is sufficient to cause strong tetramerization by creating favorable contacts with more ancient residues on the opposing subunit. These surface substitutions markedly reduce oxygen affinity and even confer cooperativity because an ancient linkage between the oxygen binding site and the multimerization interface was already an intrinsic feature of the protein’s structure. Our findings establish that evolution can produce new complex molecular structures and functions via simple genetic mechanisms that recruit existing biophysical features into higher-level architectures. The interfaces that hold molecular complexes together typically involve sterically tight, electrostatically complementary interactions among many amino acids. Similarly, allostery and cooperativity usually depend on numerous residues that connect surfaces to active sites. The acquisition of such complicated machinery would seem to require elaborate evolutionary pathways. The classical explanation of this process, by analogy to the evolution of morphological complexity, is that multimerization conferred or enhanced beneficial functions, allowing selection to drive the many substitutions required to build and optimize new interfaces. Whether this account accurately describes the evolution of any natural molecular complex requires a detailed reconstruction of the historical steps by which it evolved. Hemoglobin (Hb) is a useful model for this purpose, because the structural mechanisms that mediate its multimeric assembly, cooperative oxygen binding, and allosteric regulation are well established. Moreover, its subunits descend by duplication and divergence from the same ancestral proteins, so their history can be reconstructed in a single analysis. Despite considerable speculation, virtually nothing is known about the evolutionary origin of Hb’s heterotetrameric architecture and the functions that depend on it

The role of interfacial lipids in stabilizing membrane protein oligomers

Oligomerization of membrane proteins in response to lipid binding has a critical role in many cell-signalling pathways1 but is often difficult to define2 or predict3. Here we report the development of a mass spectrometry platform to determine simultaneously the presence of interfacial lipids and oligomeric stability and to uncover how lipids act as key regulators of membrane-protein association. Evaluation of oligomeric strength for a dataset of 125 α-helical oligomeric membrane proteins reveals an absence of interfacial lipids in the mass spectra of 12 membrane proteins with high oligomeric stability. For the bacterial homologue of the eukaryotic biogenic transporters (LeuT4, one of the proteins with the lowest oligomeric stability), we found a precise cohort of lipids within the dimer interface. Delipidation, mutation of lipid-binding sites or expression in cardiolipin-deficient Escherichia coli abrogated dimer formation. Molecular dynamics simulation revealed that cardiolipin acts as a bidentate ligand, bridging across subunits. Subsequently, we show that for the Vibrio splendidus sugar transporter SemiSWEET5, another protein with low oligomeric stability, cardiolipin shifts the equilibrium from monomer to functional dimer. We hypothesized that lipids are essential for dimerization of the Na+/H+ antiporter NhaA from E. coli, which has the lowest oligomeric strength, but not for the substantially more stable homologous Thermus thermophilus protein NapA. We found that lipid binding is obligatory for dimerization of NhaA, whereas NapA has adapted to form an interface that is stable without lipids. Overall, by correlating interfacial strength with the presence of interfacial lipids, we provide a rationale for understanding the role of lipids in both transient and stable interactions within a range of α-helical membrane proteins, including G-protein-coupled receptors

Sensing iron availability via the fragile [4Fe-4S] cluster of the bacterial transcriptional repressor RirA

Rhizobial iron regulator A (RirA) is a global regulator of iron homeostasis in many nitrogen-fixing Rhizobia and related species of α-proteobacteria. It belongs to the widespread Rrf2 super-family of transcriptional regulators and features three conserved Cys residues that characterise the binding of an iron–sulfur cluster in other Rrf2 family regulators. Here we report biophysical studies demonstrating that RirA contains a [4Fe–4S] cluster, and that this form of the protein binds RirA-regulated DNA, consistent with its function as a repressor of expression of many genes involved in iron uptake. Under low iron conditions, [4Fe–4S] RirA undergoes a cluster conversion reaction resulting in a [2Fe–2S] form, which exhibits much lower affinity for DNA. Under prolonged low iron conditions, the [2Fe–2S] cluster degrades to apo-RirA, which does not bind DNA and can no longer function as a repressor of the cell's iron-uptake machinery. [4Fe–4S] RirA was also found to be sensitive to O2, suggesting that both iron and O2 are important signals for iron metabolism. Consistent with this, in vivo data showed that expression of RirA-regulated genes is also affected by O2. These data lead us to propose a novel regulatory model for iron homeostasis, in which RirA senses iron via the incorporation of a fragile iron–sulfur cluster that is sensitive to iron and O2 concentrations

Geometrical principles of homomeric β-barrels and β-helices: Application to modeling amyloid protofilaments

Examples of homomeric β-helices and β-barrels have recently emerged. Here we generalise the theory for the shear number in β-barrels to encompass β-helices and homomeric structures. We introduce the concept of the “β-strip”, the set of parallel or antiparallel neighbouring strands, from which the whole helix can be generated giving it n-fold rotational symmetry. In this context the shear number is interpreted as the sum around the helix of the fixed register shift between neighbouring identical β-strips. Using this approach we have derived relationships between helical width, pitch, angle between strand direction and helical axis, mass per length, register shift, and number of strands. The validity and unifying power of the method is demonstrated with known structures including α-haemolysin, T4 phage spike, cylindrin, and the HET-s(218-289) prion. From reported dimensions measured by X-ray fibre diffraction on amyloid fibrils the relationships can be used to predict the register shift and the number of strands within amyloid protofilaments. This was used to construct models of transthyretin and Alzheimer β(40) amyloid protofilaments that comprise a single strip of in-register β-strands folded into a “β-strip helix”. Results suggest both stabilisation of an individual β-strip helix as well as growth by addition of further β-strip helices involves the same pair of sequence segments associating with β-sheet hydrogen bonding at the same register shift. This association would be aided by a repeat sequence. Hence understanding of how the register shift (as the distance between repeat sequences) relates to helical dimensions, will be useful for nanotube design

Mechanisms of iron- and O2-sensing by the [4Fe-4S] cluster of the global iron regulator RirA

RirA is a global regulator of iron homeostasis in Rhizobium and related α-proteobacteria. In its [4Fe-4S] cluster-bound form it represses iron uptake by binding to IRO Box sequences upstream of RirA-regulated genes. Under low iron and/or aerobic conditions, [4Fe-4S] RirA undergoes cluster conversion/degradation to apo-RirA, which can no longer bind IRO Box sequences. Here, we apply time-resolved mass spectrometry and electron paramagnetic resonance spectroscopy to determine how the RirA cluster senses iron and O2. The data indicate that the key iron-sensing step is the O2-independent, reversible dissociation of Fe2+ from [4Fe-4S]2+ to form [3Fe-4S]0. The dissociation constant for this process was determined as Kd = ~3 µM, which is consistent with the sensing of ‘free’ iron in the cytoplasm. O2-sensing occurs through enhanced cluster degradation under aerobic conditions, via O2-mediated oxidation of the [3Fe-4S]0 intermediate to form [3Fe-4S]1+. This work provides a detailed mechanistic/functional view of an iron-responsive regulator

A growing toolbox of techniques for studying β-barrel outer membrane protein folding and biogenesis

Great strides into understanding protein folding have been made since he seminal work of Anfinsen over 40 years ago, but progress in the study of membrane protein folding has lagged behind that of their water soluble counterparts. Researchers in these fields continue to turn to more advanced techniques such as NMR, mass spectrometry, molecular dynamics (MD) and single molecule methods to interrogate how proteins fold. Our understanding of β-barrel outer membrane protein (OMP) folding has benefited from these advances in the last decade. This class of proteins must traverse the periplasm and then insert into an asymmetric lipid membrane in the absence of a chemical energy source. In this review we discuss old, new and emerging techniques used to examine the process of OMP folding and biogenesis in vitro and describe some of the insights and new questions these techniques have revealed

Construction of Insulin 18-mer Nanoassemblies Driven by Coordination to Iron(II) and Zinc(II) Ions at Distinct Sites

Controlled self-assembly (SA) of proteins offers the possibility to tune their properties or to create new materials. Herein, we present the synthesis of a modified human insulin (HI) with two distinct metal-ion binding sites, one native, the other abiotic, enabling hierarchical SA through coordination with two different metal ions. Selective attachment of an abiotic 2,2'-bipyridine (bipy) ligand to HI, yielding HI-bipy, enabled Zn(II) -binding hexamers to SA into trimers of hexamers, [[HI-bipy]6 ]3 , driven by octahedral coordination to a Fe(II) ion. The structures were studied in solution by small-angle X-ray scattering and on surfaces with AFM. The abiotic metal ligand had a higher affinity for Fe(II) than Zn(II) ions, enabling control of the hexamer formation with Zn(II) and the formation of trimers of hexamers with Fe(II) ions. This precise control of protein SA to give oligomers of oligomers provides nanoscale structures with potential applications in nanomedicine