-
Current Opinion in Structural Biology Aug 2013Molecular self-assembly offers a means by which sophisticated materials can be constructed with unparalleled precision. Designing self-assembling protein structures is... (Review)
Review
Molecular self-assembly offers a means by which sophisticated materials can be constructed with unparalleled precision. Designing self-assembling protein structures is of particular interest as a result of the unique functional capabilities of proteins. Custom-designed protein materials could lead to new possibilities in therapeutics, bioenergy, and materials science. Although the field was long hampered by the challenges involved in designing such complex molecules, novel approaches and computational tools have recently led to remarkable progress. Here we review recent design studies in the context of three fundamental aspects of self-assembling materials: subunit organization, subunit interactions, and regulation of assembly.
Topics: Models, Molecular; Nanostructures; Protein Conformation; Protein Engineering; Protein Folding; Protein Subunits
PubMed: 23827813
DOI: 10.1016/j.sbi.2013.06.002 -
Journal of Molecular Modeling Nov 2010The distinguishing property of Sm protein associations is very high stability. In order to understand this property, we analyzed the interfaces and compared the...
The distinguishing property of Sm protein associations is very high stability. In order to understand this property, we analyzed the interfaces and compared the properties of Sm protein interfaces with those of a test set, the Binding Interface Database (BID). The comparison revealed that the main differences between the interfaces of Sm proteins and those of the BID set are the content of charged residues, the coordination numbers of the residues, knowledge-based pair potentials, and the conservation scores of hot spots. In Sm proteins, the interfaces have more hydrophobic and fewer charged residues than the surfaces, which is also the case for the BID test set and other proteins. However, in the interfaces, the content of charged residues in Sm proteins (26%) is substantially larger than that in the BID set (22%). Hot spots are residues that make up a small fraction of the interfaces, but they contribute most of the binding energy. These residues are critical to protein-protein interactions. Analyses of knowledge-based pair potentials of hot spot and non-hot spot residues in Sm proteins show that they are significantly different; their mean values are 31.5 and 11.3, respectively. In the BID set, this difference is smaller; in this case, the mean values for hot spot and non-hot spot residues are 20.7 and 12.4, respectively. Hence, the pair potentials of hot spots differ significantly for the Sm and BID data sets. In the interfaces of Sm proteins, the amino acids are tightly packed, and the coordination numbers are larger in Sm proteins than in the BID set for both hot spots and non-hot spots. At the same time, the coordination numbers are higher for hot spots; the average coordination number of the hot spot residues in Sm proteins is 7.7, while it is 6.1 for the non-hot spot residues. The difference in the calculated average conservation score for hot spots and non-hot spots in Sm proteins is significantly larger than it is in the BID set. In Sm proteins, the average conservation score for the hot spots is 7.4. Hot spots are surrounded by residues that are moderately conserved (5.9). The average conservation score for the other interface residues is 5.6. The conservation scores in the BID set do not show a significant distinction between hot and non-hot spots: the mean values for hot and non-hot spot residues are 5.5 and 5.2, respectively. These data show that structurally conserved residues and hot spots are significantly correlated in Sm proteins.
Topics: Amino Acids; Animals; Conserved Sequence; Databases, Protein; Humans; Hydrophobic and Hydrophilic Interactions; Protein Subunits; snRNP Core Proteins
PubMed: 20652820
DOI: 10.1007/s00894-010-0787-4 -
Biochemistry Jun 2014Nitrile hydratases (NHases) possess a mononuclear iron or cobalt cofactor whose coordination environment includes rare post-translationally oxidized cysteine sulfenic...
Nitrile hydratases (NHases) possess a mononuclear iron or cobalt cofactor whose coordination environment includes rare post-translationally oxidized cysteine sulfenic and sulfinic acid ligands. This cofactor is located in the α-subunit at the interfacial active site of the heterodimeric enzyme. Unlike canonical NHases, toyocamycin nitrile hydratase (TNHase) from Streptomyces rimosus is a unique three-subunit member of this family involved in the biosynthesis of pyrrolopyrimidine antibiotics. The subunits of TNHase are homologous to the α- and β-subunits of prototypical NHases. Herein we report the expression, purification, and characterization of the α-subunit of TNHase. The UV-visible, EPR, and mass spectra of the α-subunit TNHase provide evidence that this subunit alone is capable of synthesizing the active site complex with full post-translational modifications. Remarkably, the isolated post-translationally modified α-subunit is also catalytically active with the natural substrate, toyocamycin, as well as the niacin precursor 3-cyanopyridine. Comparisons of the steady state kinetic parameters of the single subunit variant to the heterotrimeric protein clearly show that the additional subunits impart substrate specificity and catalytic efficiency. We conclude that the α-subunit is the minimal sequence needed for nitrile hydration providing a simplified scaffold to study the mechanism and post-translational modification of this important class of catalysts.
Topics: Actinomycetales; Bacterial Proteins; Catalysis; Hydro-Lyases; Kinetics; Protein Processing, Post-Translational; Protein Subunits; Pyridines; Recombinant Proteins
PubMed: 24914472
DOI: 10.1021/bi500260j -
Cell Reports Apr 2024Fatalska et al. use an interdisciplinary strategy to elucidate how an intrinsically disordered regulatory subunit of protein phosphatase 1 binds trimeric eIF2 and...
Fatalska et al. use an interdisciplinary strategy to elucidate how an intrinsically disordered regulatory subunit of protein phosphatase 1 binds trimeric eIF2 and positions the phosphatase-substrate complex for dephosphorylation. As validation, they show that a disease mutation abolishes the interaction.
Topics: Protein Phosphatase 1; Humans; Eukaryotic Initiation Factor-2; Intrinsically Disordered Proteins; Protein Binding; Phosphorylation; Protein Subunits; Mutation
PubMed: 38573854
DOI: 10.1016/j.celrep.2024.114011 -
Biochemical and Biophysical Research... May 2006Ribonuclease P (RNase P) is a ribonucleoprotein complex involved in the processing of the 5' leader sequence of precursor tRNA. We previously found that the...
Ribonuclease P (RNase P) is a ribonucleoprotein complex involved in the processing of the 5' leader sequence of precursor tRNA. We previously found that the reconstituted particle (RP) composed of RNase P RNA and four proteins (Ph1481p, Ph1601p, Ph1771p, and Ph1877p) in the hyperthermophilic archaeon Pyrococcus horikoshii OT3 exhibited the RNase P activity, but had a lower optimal temperature (around at 55 degrees C), as compared with 70 degrees C of the authentic RNase P from P. horikoshii [Kouzuma et al., Biochem. Biophys. Res. Commun. 306 (2003) 666-673]. In the present study, we found that addition of a fifth protein Ph1496p, a putative ribosomal protein L7Ae, to RP specifically elevated the optimum temperature to about 70 degrees C comparable to that of the authentic RNase P. Characterization using gel shift assay and chemical probing localized Ph1496p binding sites on two stem-loop structures encompassing nucleotides A116-G201 and G229-C276 in P. horikoshii RNase P RNA. Moreover, the crystal structure of Ph1496p was determined at 2.0 A resolution by the molecular replacement method using ribosomal protein L7Ae from Haloarcula marismortui as a search model. Ph1496p comprises five alpha-helices and a four stranded beta-sheet. The beta-sheet is sandwiched by three helices (alpha1, alpha4, and alpha5) at one side and two helices (alpha2 and alpha3) at other side. The archaeal ribosomal protein L7Ae is known to be a triple functional protein, serving as a protein component in ribosome and ribonucleoprotein complexes, box C/D, and box H/ACA. Although we have at present no direct evidence that Ph1496p is a real protein component in the P. horikoshii RNase P, the present result may assign an RNase P protein to L7Ae as a fourth function.
Topics: Amino Acid Sequence; Archaeal Proteins; Crystallography, X-Ray; Models, Molecular; Molecular Sequence Data; Protein Subunits; Pyrococcus horikoshii; Ribonuclease P; Ribosomal Proteins; Sequence Alignment; Temperature
PubMed: 16574071
DOI: 10.1016/j.bbrc.2006.02.192 -
PLoS Biology May 2014The intrinsic flexibility of proteins allows them to undergo large conformational fluctuations in solution or upon interaction with other molecules. Proteins also...
The intrinsic flexibility of proteins allows them to undergo large conformational fluctuations in solution or upon interaction with other molecules. Proteins also commonly assemble into complexes with diverse quaternary structure arrangements. Here we investigate how the flexibility of individual protein chains influences the assembly and evolution of protein complexes. We find that flexibility appears to be particularly conducive to the formation of heterologous (i.e., asymmetric) intersubunit interfaces. This leads to a strong association between subunit flexibility and homomeric complexes with cyclic and asymmetric quaternary structure topologies. Similarly, we also observe that the more nonhomologous subunits that assemble together within a complex, the more flexible those subunits tend to be. Importantly, these findings suggest that subunit flexibility should be closely related to the evolutionary history of a complex. We confirm this by showing that evolutionarily more recent subunits are generally more flexible than evolutionarily older subunits. Finally, we investigate the very different explorations of quaternary structure space that have occurred in different evolutionary lineages. In particular, the increased flexibility of eukaryotic proteins appears to enable the assembly of heteromeric complexes with more unique components.
Topics: Animals; Apicomplexa; Arabidopsis; Bacteria; Evolution, Molecular; Fungi; Models, Molecular; Protein Multimerization; Protein Structure, Quaternary; Protein Subunits; Proteins
PubMed: 24866000
DOI: 10.1371/journal.pbio.1001870 -
International Journal of Biological... Jun 2021Protein fusion using a linker plays an important role for protein evolution. However, designing suitable linkers for protein evolution is yet challenging and...
Protein fusion using a linker plays an important role for protein evolution. However, designing suitable linkers for protein evolution is yet challenging and under-explored. To further clarify the regular pattern of suitable type of linker for fusion proteins, one nitrile hydratase (NHase) was used as a target protein and subunit fusion strategy was carried out to improve its efficient catalysis. Subunit-fused variants with three different types of linkers were constructed and characterized. All variants exhibited higher stability than that of the wild type. The longer the linker was, the higher stability NHase showed, however, too long linker affected NHase activity and expression. Among the three types of linkers, the α-helical linker seemed more suitable for NHase than flexible or rigid linkers. Though it is not clear how the linkers affecting the activity, structure analysis indicated that the stability improvement is dependent on the additional salt bridge, H-bond, and the subunit interface area increasing due to the linker insertion, among which the additional salt bridge and interface area were more important factors. The results described here may be useful for redesigning other enzymes through subunit fusion.
Topics: Biocatalysis; Catalytic Domain; Enzyme Stability; Hydro-Lyases; Kinetics; Molecular Dynamics Simulation; Protein Subunits; Recombinant Proteins; Temperature
PubMed: 33753198
DOI: 10.1016/j.ijbiomac.2021.03.103 -
Nucleic Acids Research 2007The RPM2 gene of Saccharomyces cerevisiae codes for a protein subunit of mitochondrial RNase P and has another unknown essential function. We previously demonstrated...
The RPM2 gene of Saccharomyces cerevisiae codes for a protein subunit of mitochondrial RNase P and has another unknown essential function. We previously demonstrated that Rpm2p localizes to the nucleus and acts as a transcriptional activator. Rpm2p influences the level of mRNAs that encode components of the mitochondrial import apparatus and essential mitochondrial chaperones. Evidence is presented here that Rpm2p interacts with Dcp2p, a subunit of mRNA decapping enzyme in the two-hybrid assay, and is enriched in cytoplasmic P bodies, the sites of mRNA degradation and storage in yeast and mammalian cells. When overexpressed, GFP-Rpm2p does not impact the number and size of P bodies; however, it prevents their disappearance when translation elongation is inhibited by cycloheximide. Proteasome mutants, ump1-2 and pre4-2, that bypass essential Rpm2p function, also stabilize P bodies. The stabilization of P bodies by Rpm2p may occur through reduced protein degradation since GFP-Rpm2p expressing cells have lower levels of ubiquitin. Genetic analysis revealed that overexpression of Dhh1p (a DEAD box helicase localized to P bodies) suppresses temperature-sensitive growth of the rpm2-100 mutant. Overexpression of Pab1p (a poly (A)-binding protein) also suppresses rpm2-100, suggesting that Rpm2p functions in at least two aspects of mRNA metabolism. The results presented here, and the transcriptional activation function demonstrated earlier, implicate Rpm2p as a coordinator of transcription and mRNA storage/decay in P bodies.
Topics: Cytoplasmic Structures; DEAD-box RNA Helicases; Endoribonucleases; Gene Expression Regulation, Fungal; Mitochondria; Molecular Chaperones; Peptide Chain Elongation, Translational; Poly(A)-Binding Proteins; Protein Subunits; RNA Processing, Post-Transcriptional; Ribonuclease P; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Temperature; Two-Hybrid System Techniques; Ubiquitin
PubMed: 17267405
DOI: 10.1093/nar/gkm023 -
Proceedings of the National Academy of... Oct 2020Ionotropic glutamate receptors (iGluRs) are key molecules for synaptic signaling in the central nervous system, which makes them promising drug targets. Intensive...
Ionotropic glutamate receptors (iGluRs) are key molecules for synaptic signaling in the central nervous system, which makes them promising drug targets. Intensive efforts are being devoted to the development of subunit-selective ligands, which should enable more precise pharmacologic interventions while limiting the effects on overall neuronal circuit function. However, many AMPA and kainate receptor complexes in vivo are heteromers composed of different subunits. Despite their importance, little is known about how subunit-selective ligands affect the gating of heteromeric iGluRs, namely their activation and desensitization properties. Using fast ligand application experiments, we studied the effects of competitive antagonists that block glutamate from binding at part of the four subunits. We found that UBP-310, a kainate receptor antagonist with high selectivity for GluK1 subunits, reduces the desensitization of GluK1/GluK2 heteromers and fully abolishes the desensitization of GluK1/GluK5 heteromers. This effect is mirrored by subunit-selective agonists and heteromeric receptors that contain binding-impaired subunits, as we show for both kainate and GluA2 AMPA receptors. These findings are consistent with a model in which incomplete agonist occupancy at the four receptor subunits can provide activation without inducing desensitization. However, we did not detect significant steady-state currents during UBP-310 dissociation from GluK1 homotetramers, indicating that antagonist dissociation proceeds in a nonuniform and cooperativity-driven manner, which disfavors nondesensitizing occupancy states. Besides providing mechanistic insights, these results have direct implications for the use of subunit-selective antagonists in neuroscience research and envisioned therapeutic interventions.
Topics: Dimerization; HEK293 Cells; Humans; Ligands; Protein Subunits; Receptors, Ionotropic Glutamate
PubMed: 32999066
DOI: 10.1073/pnas.2007471117 -
British Journal of Pharmacology Feb 2014Although the stoichiometry of the major synaptic αβγ subunit-containing GABAA receptors has consensus support for 2α:2β:1γ, a clear view of the stoichiometry of...
BACKGROUND AND PURPOSE
Although the stoichiometry of the major synaptic αβγ subunit-containing GABAA receptors has consensus support for 2α:2β:1γ, a clear view of the stoichiometry of extrasynaptic receptors containing δ subunits has remained elusive. Here we examine the subunit stoichiometry of recombinant α4β3δ receptors using a reporter mutation and a functional electrophysiological approach.
EXPERIMENTAL APPROACH
Using site-directed mutagenesis, we inserted a highly characterized 9' serine to leucine mutation into the second transmembrane (M2) region of α4, β3 and δ subunits that increases receptor sensitivity to GABA. Whole-cell, GABA-activated currents were recorded from HEK-293 cells co-expressing different combinations of wild-type (WT) and/or mutant α4(L297S), β3(L284S) and δ(L288S) subunits.
KEY RESULTS
Recombinant receptors containing one or more mutant subunits showed increased GABA sensitivity relative to WT receptors by approximately fourfold, independent of the subunit class (α, β or δ) carrying the mutation. GABA dose-response curves of cells co-expressing WT subunits with their respective L9'S mutants exhibited multiple components, with the number of discernible components enabling a subunit stoichiometry of 2α, 2β and 1δ to be deduced for α4β3δ receptors. Varying the cDNA transfection ratio by 10-fold had no significant effect on the number of incorporated δ subunits.
CONCLUSIONS AND IMPLICATIONS
Subunit stoichiometry is an important determinant of GABAA receptor function and pharmacology, and δ subunit-containing receptors are important mediators of tonic inhibition in several brain regions. Here we demonstrate a preferred subunit stoichiometry for α4β3δ receptors of 2α, 2β and 1δ.
Topics: Amino Acid Sequence; HEK293 Cells; Humans; Molecular Sequence Data; Mutation; Protein Subunits; Receptors, GABA-A; Sequence Alignment; gamma-Aminobutyric Acid
PubMed: 24206220
DOI: 10.1111/bph.12514