scholarly journals Discovery of a carbonic anhydrase-Rubisco supercomplex within the alpha-carboxysome

2021 ◽  
Author(s):  
Cecilia Blikstad ◽  
Eli J Dugan ◽  
Thomas G Laughlin ◽  
Mira D Liu ◽  
Sophie R Shoemaker ◽  
...  
Keyword(s):  
Carbonic Anhydrase ◽  
Binding Site ◽  
Self Assembly ◽  
Co2 Concentration ◽  
Scaffolding Protein ◽  
Structural Basis ◽  
Intrinsically Disordered ◽  
Shell Proteins ◽  
Network Of Hydrogen Bonds ◽  
Halothiobacillus Neapolitanus

Carboxysomes are proteinaceous organelles that encapsulate key enzymes of CO2 fixation, Rubisco and carbonic anhydrase, and are the centerpiece of the bacterial CO2 concentrating mechanism (CCM). In the CCM, actively accumulated cytosolic bicarbonate diffuses into the carboxysome and is converted to CO2 by carbonic anhydrase, producing a high CO2 concentration near Rubisco and ensuring efficient carboxylation. Self-assembly of the α-carboxysome is orchestrated by the intrinsically disordered scaffolding protein, CsoS2, which interacts with both Rubisco and carboxysomal shell proteins, but it is unknown how CsoSCA, the carbonic anhydrase, is incorporated into the α-carboxysome. Here, we present the structural basis of carbonic anhydrase encapsulation into α-carboxysomes from Halothiobacillus neapolitanus. We find that CsoSCA interacts directly with Rubisco via an intrinsically disordered N-terminal domain. A 1.98 Å single-particle cryo-electron microscopy structure of Rubisco in complex with this peptide reveals that CsoSCA binding is predominantly mediated by a network of hydrogen bonds. CsoSCAs binding site overlaps with that of CsoS2 but the two proteins utilize substantially different motifs and modes of binding, revealing a plasticity of the Rubisco binding site. Our results advance the understanding of biogenesis of carboxysomes and highlights the importance of Rubisco, not only as an enzyme, but also as a hub protein central for assembling supercomplexes.

Reading the phosphorylation code: binding of the 14-3-3 protein to multivalent client phosphoproteins

2020 ◽  
Vol 477 (7) ◽  
pp. 1219-1225 ◽  
Author(s):  
Nikolai N. Sluchanko
Keyword(s):  
Protein Function ◽  
Intrinsically Disordered Protein ◽  
Structural Basis ◽  
Major Protein ◽  
Post Translational Modifications ◽  
Protein Protein Interaction ◽  
Intrinsically Disordered ◽  
Biochemical Journal ◽  
Multiple Binding ◽  
And Control

Many major protein–protein interaction networks are maintained by ‘hub’ proteins with multiple binding partners, where interactions are often facilitated by intrinsically disordered protein regions that undergo post-translational modifications, such as phosphorylation. Phosphorylation can directly affect protein function and control recognition by proteins that ‘read’ the phosphorylation code, re-wiring the interactome. The eukaryotic 14-3-3 proteins recognizing multiple phosphoproteins nicely exemplify these concepts. Although recent studies established the biochemical and structural basis for the interaction of the 14-3-3 dimers with several phosphorylated clients, understanding their assembly with partners phosphorylated at multiple sites represents a challenge. Suboptimal sequence context around the phosphorylated residue may reduce binding affinity, resulting in quantitative differences for distinct phosphorylation sites, making hierarchy and priority in their binding rather uncertain. Recently, Stevers et al. [Biochemical Journal (2017) 474: 1273–1287] undertook a remarkable attempt to untangle the mechanism of 14-3-3 dimer binding to leucine-rich repeat kinase 2 (LRRK2) that contains multiple candidate 14-3-3-binding sites and is mutated in Parkinson's disease. By using the protein-peptide binding approach, the authors systematically analyzed affinities for a set of LRRK2 phosphopeptides, alone or in combination, to a 14-3-3 protein and determined crystal structures for 14-3-3 complexes with selected phosphopeptides. This study addresses a long-standing question in the 14-3-3 biology, unearthing a range of important details that are relevant for understanding binding mechanisms of other polyvalent proteins.

scholarly journals Conformational Ensembles of an Intrinsically Disordered Protein pKID with and without a KIX Domain in Explicit Solvent Investigated by All-Atom Multicanonical Molecular Dynamics

Biomolecules ◽  
2012 ◽  
Vol 2 (1) ◽  
pp. 104-121 ◽  
Author(s):  
Koji Umezawa ◽  
Jinzen Ikebe ◽  
Mitsunori Takano ◽  
Haruki Nakamura ◽  
Junichi Higo
Keyword(s):  
Molecular Dynamics ◽  
Free Energy ◽  
Binding Site ◽  
Complex Structure ◽  
Intrinsically Disordered Protein ◽  
Bound State ◽  
Conformational Ensembles ◽  
Intrinsically Disordered ◽  
Dynamics Simulations ◽  
High Flexibility

The phosphorylated kinase-inducible activation domain (pKID) adopts a helix–loop–helix structure upon binding to its partner KIX, although it is unstructured in the unbound state. The N-terminal and C-terminal regions of pKID, which adopt helices in the complex, are called, respectively, αA and αB. We performed all-atom multicanonical molecular dynamics simulations of pKID with and without KIX in explicit solvents to generate conformational ensembles. Although the unbound pKID was disordered overall, αA and αB exhibited a nascent helix propensity; the propensity of αA was stronger than that of αB, which agrees with experimental results. In the bound state, the free-energy landscape of αB involved two low free-energy fractions: native-like and non-native fractions. This result suggests that αB folds according to the induced-fit mechanism. The αB-helix direction was well aligned as in the NMR complex structure, although the αA helix exhibited high flexibility. These results also agree quantitatively with experimental observations. We have detected that the αB helix can bind to another site of KIX, to which another protein MLL also binds with the adopting helix. Consequently, MLL can facilitate pKID binding to the pKID-binding site by blocking the MLL-binding site. This also supports experimentally obtained results.

scholarly journals In Vitro Assembly of the T=13 Procapsid of Bacteriophage T5 with Its Scaffolding Domain

2010 ◽  
Vol 84 (18) ◽  
pp. 9350-9358 ◽  
Author(s):  
Alexis Huet ◽  
James F. Conway ◽  
Lucienne Letellier ◽  
Pascale Boulanger
Keyword(s):  
Self Assembly ◽  
Scaffolding Protein ◽  
Accessory Proteins ◽  
Conformational Diversity ◽  
In Vitro Assembly ◽  
20 Nm ◽  
Head Protein ◽  
Bacteriophage T5 ◽  
Icosahedral Capsid

ABSTRACT The Siphoviridae coliphage T5 differs from other members of this family by the size of its genome (121 kbp) and by its large icosahedral capsid (90 nm), which is organized with T=13 geometry. T5 does not encode a separate scaffolding protein, but its head protein, pb8, contains a 159-residue aminoterminal scaffolding domain (Δ domain) that is the mature capsid. We have deciphered the early events of T5 shell assembly starting from purified pb8 with its Δ domain (pb8p). The self assembly of pb8p is regulated by salt conditions and leads to structures with distinct morphologies. Expanded tubes are formed in the presence of NaCl, whereas Ca2+ promotes the association of pb8p into contracted tubes and procapsids. Procapsids display an angular organization and 20-nm-long internal radial structures identified as the Δ domain. The T5 head maturation protease pb11 specifically cleaves the Δ domain of contracted and expanded tubes. Ca2+ is not required for proteolytic activity but for the organization of the Δ domain. Taken together, these data indicate that pb8p carries all of the information in its primary sequence to assemble in vitro without the requirement of the portal and accessory proteins. Furthermore, Ca2+ plays a key role in introducing the conformational diversity that permits the formation of a stable procapsid. Phage T5 is the first example of a viral capsid consisting of quasi-equivalent hexamers and pentamers whose assembly can be carried out in vitro, starting from the major head protein with its scaffolding domain, and whose endpoint is an icosahedral T=13 particle.

scholarly journals Structural Basis for Inhibition of Protein-tyrosine Phosphatase 1B by Isothiazolidinone Heterocyclic Phosphonate Mimetics

2006 ◽  
Vol 281 (43) ◽  
pp. 32784-32795 ◽  
Author(s):  
Paul J. Ala ◽  
Lucie Gonneville ◽  
Milton C. Hillman ◽  
Mary Becker-Pasha ◽  
Min Wei ◽  
...  
Keyword(s):  
Protein Tyrosine Phosphatase ◽  
Tyrosine Phosphatase ◽  
Structural Basis ◽  
Side Chain ◽  
Protein Tyrosine Phosphatase 1B ◽  
Phosphate Binding ◽  
Protein Tyrosine ◽  
Starting Point ◽  
Network Of Hydrogen Bonds ◽  
Orthogonal Orientation

Crystal structures of protein-tyrosine phosphatase 1B in complex with compounds bearing a novel isothiazolidinone (IZD) heterocyclic phosphonate mimetic reveal that the heterocycle is highly complementary to the catalytic pocket of the protein. The heterocycle participates in an extensive network of hydrogen bonds with the backbone of the phosphate-binding loop, Phe182 of the flap, and the side chain of Arg221. When substituted with a phenol, the small inhibitor induces the closed conformation of the protein and displaces all waters in the catalytic pocket. Saturated IZD-containing peptides are more potent inhibitors than unsaturated analogs because the IZD heterocycle and phenyl ring directly attached to it bind in a nearly orthogonal orientation with respect to each other, a conformation that is close to the energy minimum of the saturated IZD-phenyl moiety. These results explain why the heterocycle is a potent phosphonate mimetic and an ideal starting point for designing small nonpeptidic inhibitors.

scholarly journals Crystal structure of human carbonic anhydrase II at 1.95 Å resolution in complex with 667-coumate, a novel anti-cancer agent

2005 ◽  
Vol 385 (3) ◽  
pp. 715-720 ◽  
Author(s):  
Matthew D. LLOYD ◽  
Richard L. PEDERICK ◽  
Ramanathan NATESH ◽  
L. W. Lawrence WOO ◽  
Atul PUROHIT ◽  
...  
Keyword(s):  
Carbonic Anhydrase ◽  
Phase 1 ◽  
Structural Basis ◽  
Pharmacokinetic Studies ◽  
Reversible Binding ◽  
X Ray Crystallography ◽  
Hydrophobic Binding ◽  
Cancer Agent

CA (carbonic anhydrase) catalyses the reversible hydration of carbon dioxide into bicarbonate, and at least 14 isoforms have been identified in vertebrates. The role of CA type II in maintaining the fluid and pH balance has made it an attractive drug target for the treatment of glaucoma and cancer. 667-Coumate is a potent inhibitor of the novel oncology target steroid sulphatase and is currently in Phase 1 clinical trials for hormone-dependent breast cancer. It also inhibits CA II in vitro. In the present study, CA II was crystallized with 667-coumate and the structure was determined by X-ray crystallography at 1.95 Å (1 Å=0.1 nm) resolution. The structure reported here is the first for an inhibitor based on a coumarin ring and shows ligation of the sulphamate group to the active-site zinc at 2.15 Å through a nitrogen anion. The first two rings of the coumarin moiety are bound within the hydrophobic binding site of CA II. Important residues contributing to binding include Val-121, Phe-131, Val-135, Leu-141, Leu-198 and Pro-202. The third seven-membered ring is more mobile and is located in the channel leading to the surface of the enzyme. Pharmacokinetic studies show enhanced stability of 667-coumate in vivo and this has been ascribed to binding of CA II in erythrocytes. This result provides a structural basis for the stabilization and long half-life of 667-coumate in blood compared with its rapid disappearance in plasma, and suggests that reversible binding of inhibitors to CA may be a general method of delivering this type of labile drug.

scholarly journals Human Group C Rotavirus VP8*s Recognize Type A Histo-Blood Group Antigens as Ligands

2018 ◽  
Vol 92 (11) ◽  
Author(s):  
Xiaoman Sun ◽  
Lihong Wang ◽  
Jianxun Qi ◽  
Dandi Li ◽  
Mengxuan Wang ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
Blood Group ◽  
Specific Interaction ◽  
Type A ◽  
Host Susceptibility ◽  
Structural Basis ◽  
Receptor Interaction ◽  
Blood Group Antigens ◽  
Group Species

ABSTRACTGroup/species C rotaviruses (RVCs) have been identified as important pathogens of acute gastroenteritis (AGE) in children, family-based outbreaks, as well as animal infections. However, little is known regarding their host-specific interaction, infection, and pathogenesis. In this study, we performed serial studies to characterize the function and structural features of a human G4P[2] RVC VP8* that is responsible for the host receptor interaction. Glycan microarrays demonstrated that the human RVC VP8* recognizes type A histo-blood group antigens (HBGAs), which was confirmed by synthetic glycan-/saliva-based binding assays and hemagglutination of red blood cells, establishing a paradigm of RVC VP8*-glycan interactions. Furthermore, the high-resolution crystal structure of the human RVC VP8* was solved, showing a typical galectin-like structure consisting of two β-sheets but with significant differences from cogent proteins of group A rotaviruses (RVAs). The VP8* in complex with a type A trisaccharide displays a novel ligand binding site that consists of a particular set of amino acid residues of the C-D, G-H, and K-L loops. RVC VP8* interacts with type A HBGAs through a unique mechanism compared with that used by RVAs. Our findings shed light on the host-virus interaction and the coevolution of RVCs and will facilitate the development of specific antivirals and vaccines.IMPORTANCEGroup/species C rotaviruses (RVCs), members ofReoviridaefamily, infect both humans and animals, but our knowledge about the host factors that control host susceptibility and specificity is rudimentary. In this work, we characterized the glycan binding specificity and structural basis of a human RVC that recognizes type A HBGAs. We found that human RVC VP8*, the rotavirus host ligand binding domain that shares only ∼15% homology with the VP8* domains of RVAs, recognizes type A HBGA at an as-yet-unknown glycan binding site through a mechanism distinct from that used by RVAs. Our new advancements provide insights into RVC-cell attachment, the critical step of virus infection, which will in turn help the development of control and prevention strategies against RVs.

scholarly journals Comprehensive understanding of acetohydroxyacid synthase inhibition by different herbicide families

2017 ◽  
Vol 114 (7) ◽  
pp. E1091-E1100 ◽  
Author(s):  
Mario D. Garcia ◽  
Amanda Nouwens ◽  
Thierry G. Lonhienne ◽  
Luke W. Guddat
Keyword(s):  
Binding Site ◽  
Crystal Structures ◽  
Kinetic Studies ◽  
Herbicidal Activity ◽  
Structural Basis ◽  
Acetohydroxyacid Synthase ◽  
Branched Chain Amino Acid ◽  
Application Rates ◽  
Herbicide Binding ◽  
Branched Chain

Five commercial herbicide families inhibit acetohydroxyacid synthase (AHAS, E.C. 2.2.1.6), which is the first enzyme in the branched-chain amino acid biosynthesis pathway. The popularity of these herbicides is due to their low application rates, high crop vs. weed selectivity, and low toxicity in animals. Here, we have determined the crystal structures of Arabidopsis thaliana AHAS in complex with two members of the pyrimidinyl-benzoate (PYB) and two members of the sulfonylamino-carbonyl-triazolinone (SCT) herbicide families, revealing the structural basis for their inhibitory activity. Bispyribac, a member of the PYBs, possesses three aromatic rings and these adopt a twisted “S”-shaped conformation when bound to A. thaliana AHAS (AtAHAS) with the pyrimidinyl group inserted deepest into the herbicide binding site. The SCTs bind such that the triazolinone ring is inserted deepest into the herbicide binding site. Both compound classes fill the channel that leads to the active site, thus preventing substrate binding. The crystal structures and mass spectrometry also show that when these herbicides bind, thiamine diphosphate (ThDP) is modified. When the PYBs bind, the thiazolium ring is cleaved, but when the SCTs bind, ThDP is modified to thiamine 2-thiazolone diphosphate. Kinetic studies show that these compounds not only trigger reversible accumulative inhibition of AHAS, but also can induce inhibition linked with ThDP degradation. Here, we describe the features that contribute to the extraordinarily powerful herbicidal activity exhibited by four classes of AHAS inhibitors.

scholarly journals Structural control for the coordinated assembly into functional pathogenic type-3 secretion systems

2019 ◽  
Author(s):  
Nikolaus Goessweiner-Mohr ◽  
Vadim Kotov ◽  
Matthias J. Brunner ◽  
Julia Mayr ◽  
Jiri Wald ◽  
...  
Keyword(s):  
Self Assembly ◽  
Structural Control ◽  
Helical Structure ◽  
Structural Basis ◽  
Protein Chain ◽  
Assembly Process ◽  
Secretion Systems ◽  
Ring Assembly ◽  
Serovar Typhimurium ◽  
Type 3

AbstractFunctional injectisomes of the type-3 secretion system assemble into highly defined and stoichiometric bacterial molecular machines essential for infecting human and other eukaryotic cells. However, the mechanism that governs the regulated step-wise assembly process from the nucleation-phase, to ring-assembly, and the filamentous phase into a membrane embedded needle complex is unclear. We here report that the formation of a megadalton-sized needle complexes from Salmonella enterica serovar Typhimurium (SPI-1, Salmonella pathogenicity island-1) with proper stoichiometries is highly structurally controlled competing against the self-assembly propensity of injectisome components, leading to a highly unusual structurally-pleiotropic phenotype. The structure of the entire needle complex from pathogenic injectisomes was solved by cryo electron microscopy, focused refinements (2.5-4 Å) and co-variation analysis revealing an overall asymmetric arrangement containing cyclic, helical, and asymmetric sub-structures. The centrally located export apparatus assembles into a conical, pseudo-helical structure and provides a structural template that guides the formation of a 24-mer cyclic, surrounding ring, which then serves as a docking interface comprising three different conformations for sixteen N-terminal InvG subunits of the outer secretin ring. Unexpectedly, the secretin ring excludes the 16th protein chain at the C-terminal outer ring, resulting in a pleiotropic 16/15-mer ring and consequently to an overall 24:16/15 basal body structure. Finally, we report how the transition from the pseudo-helical export apparatus into the helical filament is structurally resolved to generate the protein secretion channel, which provides the structural basis to restrict access of unfolded effector substrates. These results highlight the diverse molecular signatures required for a highly coordinated assembly process and provide the molecular basis for understanding triggering and transport of unfolded proteins through injectisomes.

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