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Biochemistry Jul 2015Small heat shock proteins (sHSPs) make up a class of molecular chaperones broadly observed across organisms. Many sHSPs form large oligomers that undergo dynamic subunit...
Small heat shock proteins (sHSPs) make up a class of molecular chaperones broadly observed across organisms. Many sHSPs form large oligomers that undergo dynamic subunit exchange that is thought to play a role in chaperone function. Though remarkably heterogeneous, sHSP oligomers share three types of intermolecular interactions that involve all three defined regions of a sHSP: the N-terminal region (NTR), the conserved α-crystallin domain (ACD), and a C-terminal region (CTR). Here we define the structural interactions involved in incorporation of a subunit into a sHSP oligomer. We demonstrate that a minimal ACD dimer of the human sHSP, HSPB5, interacts with an HSPB5 oligomer through two types of interactions: (1) interactions with CTRs in the oligomer and (2) via exchange into and out of the dimer interface composed of two ACDs. Unexpectedly, although dimers are thought to be the fundamental building block for sHSP oligomers, our results clearly indicate that subunit exchange into and out of oligomers occurs via monomers. Using structure-based mutants, we show that incorporation of a subunit into an oligomer is predicated on recruitment of the subunit via its interaction with CTRs on an oligomer. Both the rate and extent of subunit incorporation depend on the accessibility of CTRs within an HSPB5 oligomer. We show that this mechanism also applies to formation of heterooligomeric sHSP species composed of HSPB5 and HSPB6 and is likely general among sHSPs. Finally, our observations highlight the importance of NTRs in the thermodynamic stability of sHSP oligomers.
Topics: Heat-Shock Proteins, Small; Humans; Models, Molecular; Protein Conformation; Protein Interaction Domains and Motifs; Protein Multimerization; Protein Subunits; alpha-Crystallins
PubMed: 26098708
DOI: 10.1021/acs.biochem.5b00490 -
Annual Review of Cell and Developmental... 2004Dynactin is a multisubunit protein complex that is required for most, if not all, types of cytoplasmic dynein activity in eukaryotes. Dynactin binds dynein directly and... (Review)
Review
Dynactin is a multisubunit protein complex that is required for most, if not all, types of cytoplasmic dynein activity in eukaryotes. Dynactin binds dynein directly and allows the motor to traverse the microtubule lattice over long distances. A single dynactin subunit, p150Glued, is sufficient for both activities, yet dynactin contains several other subunits that are organized into an elaborate structure. It is currently believed that the bulk of the dynactin structure participates in interactions with a wide range of cellular structures, many of which are cargoes of the dynein motor. Genetic studies verify the importance of all elements of dynactin structure to its function. Although dynein can bind some membranous cargoes independently of dynactin, establishment of a fully functional dynein-cargo link appears to depend on dynactin. In this review, I summarize what is presently known about dynactin structure, the cellular structures with which it associates, and the intermolecular interactions that underlie and regulate binding. Although the molecular details of dynactin's interactions with membranous organelles and other molecules are complex, the framework provided here is intended to distill what is presently known and to be of use to dynactin specialists and beginners alike.
Topics: Animals; Dynactin Complex; Intracellular Space; Microtubule-Associated Proteins; Protein Subunits
PubMed: 15473859
DOI: 10.1146/annurev.cellbio.20.012103.094623 -
Trends in Neurosciences Nov 2008Ionotropic receptors mediate rapid communication between neurons. These receptors are oligomers and are usually assembled from multiple subunit types. Receptors built... (Review)
Review
Ionotropic receptors mediate rapid communication between neurons. These receptors are oligomers and are usually assembled from multiple subunit types. Receptors built from different subunit combinations have distinct functional properties, such as single-channel conductances, rates of desensitization and sensitivities to activators and inactivators; they can also have different intracellular locations. Methods are now available for determining not only the subunit stoichiometry but also the subunit arrangement within ionotropic receptors. This information will inform experiments designed to understand the molecular basis of receptor assembly and function. It will also permit the modelling of potential ligand-binding sites at the interfaces between the subunits and should lead to a more rational approach to drug development.
Topics: Animals; Models, Molecular; Protein Conformation; Protein Subunits; Receptors, Neurotransmitter
PubMed: 18774187
DOI: 10.1016/j.tins.2008.08.001 -
Acta Physiologica (Oxford, England) May 2009AMP-activated protein kinase (AMPK) regulates metabolism in response to energy demand and supply. AMPK is activated in response to rises in intracellular AMP or... (Review)
Review
AMP-activated protein kinase (AMPK) regulates metabolism in response to energy demand and supply. AMPK is activated in response to rises in intracellular AMP or calcium-mediated signalling and is responsible for phosphorylating a wide variety of substrates. Recent structural studies have revealed the architecture of the alphabetagamma subunit interactions as well as the AMP binding pockets on the gamma subunit. The alpha catalytic domain (1-280) is autoinhibited by a C-terminal tail (313-335), which is proposed to interact with the small lobe of the catalytic domain by homology modelling with the MARK2 protein structure. Two direct activating drugs have been reported for AMPK, the thienopyridone compound A769662 and PTI, which may activate by distinct mechanisms.
Topics: AMP-Activated Protein Kinases; Adenosine Monophosphate; Amino Acid Sequence; Animals; Binding Sites; Enzyme Activation; Models, Molecular; Molecular Sequence Data; Molecular Structure; Protein Conformation; Protein Subunits; Signal Transduction
PubMed: 19245650
DOI: 10.1111/j.1748-1716.2009.01977.x -
FEBS Letters Dec 2006A comparative study of the 30S ribosomal subunit in the complex with protein S1 and the subunit depleted of this protein has been carried out by the hot tritium...
A comparative study of the 30S ribosomal subunit in the complex with protein S1 and the subunit depleted of this protein has been carried out by the hot tritium bombardment method. Differences in exposure of some ribosomal proteins within the 30S subunit depleted of S1 and within the 30S-S1 complex were found. It was concluded that protein S1 binds in the region of the neck of the 30S ribosomal subunit inducing a conformational change of its structure.
Topics: Electrophoresis, Gel, Two-Dimensional; Models, Molecular; Protein Binding; Protein Structure, Quaternary; Protein Subunits; Ribosomal Proteins; Thermus thermophilus
PubMed: 17150214
DOI: 10.1016/j.febslet.2006.11.036 -
International Journal of Molecular... Sep 2021Ribonuclease P (RNase P) is an important ribonucleoprotein (RNP), responsible for the maturation of the 5' end of precursor tRNAs (pre-tRNAs). In all organisms, the... (Review)
Review
Ribonuclease P (RNase P) is an important ribonucleoprotein (RNP), responsible for the maturation of the 5' end of precursor tRNAs (pre-tRNAs). In all organisms, the cleavage activity of a single phosphodiester bond adjacent to the first nucleotide of the acceptor stem is indispensable for cell viability and lies within an essential catalytic RNA subunit. Although RNase P is a ribozyme, its kinetic efficiency in vivo, as well as its structural variability and complexity throughout evolution, requires the presence of one protein subunit in bacteria to several protein partners in archaea and eukaryotes. Moreover, the existence of protein-only RNase P (PRORP) enzymes in several organisms and organelles suggests a more complex evolutionary timeline than previously thought. Recent detailed structures of bacterial, archaeal, human and mitochondrial RNase P complexes suggest that, although apparently dissimilar enzymes, they all recognize pre-tRNAs through conserved interactions. Interestingly, individual protein subunits of the human nuclear and mitochondrial holoenzymes have additional functions and contribute to a dynamic network of elaborate interactions and cellular processes. Herein, we summarize the role of each RNase P subunit with a focus on the human nuclear RNP and its putative role in flawless gene expression in light of recent structural studies.
Topics: Animals; Catalytic Domain; Humans; Kinetics; Protein Subunits; RNA Precursors; RNA, Catalytic; Ribonuclease P
PubMed: 34638646
DOI: 10.3390/ijms221910307 -
Proceedings of the National Academy of... Jul 2022Function follows form in biology, and the binding of small molecules requires proteins with pockets that match the shape of the ligand. For design of binding to...
Function follows form in biology, and the binding of small molecules requires proteins with pockets that match the shape of the ligand. For design of binding to symmetric ligands, protein homo-oligomers with matching symmetry are advantageous as each protein subunit can make identical interactions with the ligand. Here, we describe a general approach to designing hyperstable C2 symmetric proteins with pockets of diverse size and shape. We first designed repeat proteins that sample a continuum of curvatures but have low helical rise, then docked these into C2 symmetric homodimers to generate an extensive range of C2 symmetric cavities. We used this approach to design thousands of C2 symmetric homodimers, and characterized 101 of them experimentally. Of these, the geometry of 31 were confirmed by small angle X-ray scattering and 2 were shown by crystallographic analyses to be in close agreement with the computational design models. These scaffolds provide a rich set of starting points for binding a wide range of C2 symmetric compounds.
Topics: Ligands; Models, Molecular; Protein Binding; Protein Subunits
PubMed: 35862457
DOI: 10.1073/pnas.2113400119 -
Current Opinion in Structural Biology Feb 2019Ionotropic glutamate receptors in vertebrates are composed of three major subtypes - AMPA, kainate, and NMDA receptors - and mediate the majority of fast excitatory... (Review)
Review
Ionotropic glutamate receptors in vertebrates are composed of three major subtypes - AMPA, kainate, and NMDA receptors - and mediate the majority of fast excitatory neurotransmission at chemical synapses of the central nervous system. Among the three major families, native AMPA receptors function as complexes with a variety of auxiliary subunits, which in turn modulate receptor trafficking, gating, pharmacology, and permeation. Despite the long history of structure-mechanism studies using soluble receptor domains or intact yet isolated receptors, structures of AMPA receptor-auxiliary subunit complexes have not been available until recent breakthroughs in single-particle cryo-electron microscopy. Single particle cryo-EM studies have, in turn, provided new insights into the structure and organization of AMPA receptor - auxiliary protein complexes and into the molecular mechanisms of AMPA receptor activation and desensitization.
Topics: Animals; Protein Subunits; Receptors, AMPA
PubMed: 30825796
DOI: 10.1016/j.sbi.2019.01.011 -
Clinical Chemistry and Laboratory... Jun 2020Background While quantitation methods for small-molecule and tryptic peptide bottom-up mass spectrometry (MS) have been well defined, quantitation methods for top-down...
Background While quantitation methods for small-molecule and tryptic peptide bottom-up mass spectrometry (MS) have been well defined, quantitation methods for top-down or middle-up MS approaches have not been as well defined. Therapeutic monoclonal antibodies (t-mAbs) are a group of proteins that can be used to both demonstrate the advantages of top-down or middle-up detection methods over classic tryptic peptide bottom-up along with the growing need for robust quantitation strategies/software for these top-down or middle-up methods. Bottom-up proteolytic digest methods for the t-mAbs tend to suffer from challenges such as limited peptide selection due to potential interference from the polyclonal immunoglobulin background, complicated workflows, and inadequate sensitivity and specificity without laborious purification steps, and therefore have prompted the search for new detection and quantitation methods. Time-of-flight along with Orbitrap MS have recently evolved from the research and/or pharmaceutical setting into the clinical laboratory. With their superior mass measurement accuracy, resolution and scanning speeds, these are ideal platforms for top-down or middle-up characterization and quantitation. Methods We demonstrate a validated, robust, middle-up protein subunit detection and quantitation method for the IgG1 t-mAb, vedolizumab (VEDO), which takes advantage of the high resolution of the Orbitrap MS detection and quantitation software to increase specificity. Results Validated performance characteristics met pre-defined acceptance criteria with simple workflows and rapid turnaround times: characteristics necessary for implementation into a high-volume clinical MS laboratory. Conclusions While the extraction method can easily be used with other IgG1 t-mAbs, the detection and quantitation method may become an option for measurement of other proteins.
Topics: Antibodies, Monoclonal, Humanized; Humans; Mass Spectrometry; Protein Subunits; Software
PubMed: 31756160
DOI: 10.1515/cclm-2019-0862 -
Proteins Dec 2021For CASP14, we developed deep learning-based methods for predicting homo-oligomeric and hetero-oligomeric contacts and used them for oligomer modeling. To build...
For CASP14, we developed deep learning-based methods for predicting homo-oligomeric and hetero-oligomeric contacts and used them for oligomer modeling. To build structure models, we developed an oligomer structure generation method that utilizes predicted interchain contacts to guide iterative restrained minimization from random backbone structures. We supplemented this gradient-based fold-and-dock method with template-based and ab initio docking approaches using deep learning-based subunit predictions on 29 assembly targets. These methods produced oligomer models with summed Z-scores 5.5 units higher than the next best group, with the fold-and-dock method having the best relative performance. Over the eight targets for which this method was used, the best of the five submitted models had average oligomer TM-score of 0.71 (average oligomer TM-score of the next best group: 0.64), and explicit modeling of inter-subunit interactions improved modeling of six out of 40 individual domains (ΔGDT-TS > 2.0).
Topics: Computational Biology; Databases, Protein; Deep Learning; Models, Molecular; Protein Binding; Protein Conformation; Protein Subunits; Proteins; Sequence Analysis, Protein; Software
PubMed: 34324224
DOI: 10.1002/prot.26197