Structure of the bacterial flagellar hook cap provides insights into a hook assembly mechanism

AbstractAssembly of bacterial flagellar hook requires FlgD, a protein known to form the hook cap. Symmetry mismatch between the hook and the hook cap is believed to drive efficient assembly of the hook in a way similar to the filament cap helping filament assembly. However, the hook cap dependent mechanism of hook assembly has remained poorly understood. Here, we report the crystal structure of the hook cap composed of five subunits of FlgD from Salmonella enterica at 3.3 Å resolution. The pentameric structure of the hook cap is divided into two parts: a stalk region composed of five N-terminal domains; and a petal region containing five C-terminal domains. Biochemical and genetic analyses show that the N-terminal domains of the hook cap is essential for the hook-capping function, and the structure now clearly reveals why. A plausible hook assembly mechanism promoted by the hook cap is proposed based on the structure.

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Polylysine induces an antiparallel actin dimer that nucleates filament assembly. Crystal structure at 3.5-Å resolution.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(20)74514-x ◽

2002 ◽

Vol 277 (36) ◽

pp. 33529

Author(s):

Michael R. Bubb ◽

Lakshmanan Govindasamy ◽

Elena G. Yarmola ◽

Sergey M. Vorobiev ◽

Steven C. Almo ◽

...

Keyword(s):

Crystal Structure ◽

Filament Assembly

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Addressing the Molecular Mechanism of Longitudinal Lamin Assembly Using Chimeric Fusions

Cells ◽

10.3390/cells9071633 ◽

2020 ◽

Vol 9 (7) ◽

pp. 1633 ◽

Cited By ~ 2

Author(s):

Giel Stalmans ◽

Anastasia V. Lilina ◽

Pieter-Jan Vermeire ◽

Jan Fiala ◽

Petr Novák ◽

...

Keyword(s):

Cell Nucleus ◽

Molecular Model ◽

Coiled Coil ◽

Cross Linking ◽

Specific Interactions ◽

Molecular Architecture ◽

Filament Assembly ◽

Assembly Mechanism ◽

Recombinant Fragments ◽

Chemical Cross Linking

The molecular architecture and assembly mechanism of intermediate filaments have been enigmatic for decades. Among those, lamin filaments are of particular interest due to their universal role in cell nucleus and numerous disease-related mutations. Filament assembly is driven by specific interactions of the elementary dimers, which consist of the central coiled-coil rod domain flanked by non-helical head and tail domains. We aimed to investigate the longitudinal ‘head-to-tail’ interaction of lamin dimers (the so-called ACN interaction), which is crucial for filament assembly. To this end, we prepared a series of recombinant fragments of human lamin A centred around the N- and C-termini of the rod. The fragments were stabilized by fusions to heterologous capping motifs which provide for a correct formation of parallel, in-register coiled-coil dimers. As a result, we established crystal structures of two N-terminal fragments one of which highlights the propensity of the coiled-coil to open up, and one C-terminal rod fragment. Additional studies highlighted the capacity of such N- and C-terminal fragments to form specific complexes in solution, which were further characterized using chemical cross-linking. These data yielded a molecular model of the ACN complex which features a 6.5 nm overlap of the rod ends.

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Self-assembling triazolophanes: from croissants through donuts to spherical vesicles

Chemical Communications ◽

10.1039/c4cc09587a ◽

2015 ◽

Vol 51 (32) ◽

pp. 6905-6908 ◽

Cited By ~ 14

Author(s):

V. Haridas ◽

Appa Rao Sapala ◽

Jerry P. Jasinski

Keyword(s):

Crystal Structure ◽

Self Assembly ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

X Ray ◽

Self Assembling ◽

Assembly Mechanism ◽

Spherical Vesicles

A detailed ultramicroscopic analysis of three novel triazolophanes demonstrated a hierarchical self-assembly mechanism. These macrocycles self-assemble in a concentration dependent manner to hemi-toroids, toroids and finally to vesicles. The finding was supported by ultramicroscopy and X-ray crystal structure studies.

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Crystal structure of domains 3 and 4 of rat CD4: relation to the NH2-terminal domains

Science ◽

10.1126/science.8493535 ◽

1993 ◽

Vol 260 (5110) ◽

pp. 979-983 ◽

Cited By ~ 100

Author(s):

R. Brady ◽

E. Dodson ◽

G. Dodson ◽

G Lange ◽

S. Davis ◽

...

Keyword(s):

Crystal Structure ◽

Terminal Domains

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Cryo-EM Structures of CTP Synthase Filaments Reveal Mechanism of pH-Sensitive Assembly During Budding Yeast Starvation

10.1101/2021.08.25.457724 ◽

2021 ◽

Author(s):

Jesse M Hansen ◽

Avital Horowitz ◽

Eric M Lynch ◽

Daniel P Farrell ◽

Joel Quispe ◽

...

Keyword(s):

Metabolic Enzymes ◽

Ph Sensitive ◽

Starvation Response ◽

Filament Assembly ◽

Assembly Mechanism ◽

Ctp Synthase ◽

Filament Bundles ◽

Cytoplasmic Acidification

ABSTRACTMany metabolic enzymes self-assemble into micron-scale filaments to organize and regulate metabolism. The appearance of these assemblies often coincides with large metabolic changes as in development, cancer, and stress. Yeast undergo cytoplasmic acidification upon starvation, triggering the assembly of many metabolic enzymes into filaments. However, it is unclear how these filaments assemble at the molecular level and what their role is in the yeast starvation response. CTP Synthase (CTPS) assembles into metabolic filaments across many species. Here, we characterize in vitro polymerization and investigate in vivo consequences of CTPS assembly in yeast. Cryo-EM structures reveal a pH-sensitive assembly mechanism and highly ordered filament bundles that stabilize an inactive state of the enzyme, features unique to yeast CTPS. Disruption of filaments in cells with non-assembly or hyper-assembly mutations decreases growth rate, reflecting the importance of regulated CTPS filament assembly in homeotstasis.

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Engineering and crystal structure of a monomeric FLT3 ligand variant

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x21003289 ◽

2021 ◽

Vol 77 (4) ◽

pp. 121-127

Author(s):

Erwin Pannecoucke ◽

Laurens Raes ◽

Savvas N. Savvides

Keyword(s):

Crystal Structure ◽

Tyrosine Kinases ◽

Structural Studies ◽

Flt3 Ligand ◽

Class Iii ◽

Wild Type ◽

Dimer Interface ◽

Assembly Mechanism ◽

Receptor Interactions ◽

Dimerization Interface

The overarching paradigm for the activation of class III and V receptor tyrosine kinases (RTKs) prescribes cytokine-mediated dimerization of the receptor ectodomains and homotypic receptor–receptor interactions. However, structural studies have shown that the hematopoietic receptor FLT3, a class III RTK, does not appear to engage in such receptor–receptor contacts, despite its efficient dimerization by dimeric FLT3 ligand (FL). As part of efforts to better understand the intricacies of FLT3 activation, we sought to engineer a monomeric FL. It was found that a Leu27Asp substitution at the dimer interface of the cytokine led to a stable monomeric cytokine (FLL27D) without abrogation of receptor binding. The crystal structure of FLL27D at 1.65 Å resolution revealed that the introduced point mutation led to shielding of the hydrophobic footprint of the dimerization interface in wild-type FL without affecting the conformation of the FLT3 binding site. Thus, FLL27D can serve as a monomeric FL variant to further interrogate the assembly mechanism of extracellular complexes of FLT3 in physiology and disease.

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The FlhA linker mediates flagellar protein export switching during flagellar assembly

10.1101/2020.09.09.290478 ◽

2020 ◽

Author(s):

Yumi Inoue ◽

Mamoru Kida ◽

Miki Kinoshita ◽

Norihiro Takekawa ◽

Keiichi Namba ◽

...

Keyword(s):

Crystal Structure ◽

Binding Site ◽

Cytoplasmic Domain ◽

Ring Structure ◽

Protein Export ◽

Filament Assembly ◽

Flagellar Assembly ◽

Chaperone Binding ◽

Flagellar Protein ◽

Filament Type

AbstractThe flagellar protein export apparatus switches export specificity from hook-type to filament-type upon completion of hook assembly, thereby initiating filament assembly at the hook tip. The C-terminal cytoplasmic domain of FlhA (FlhAC) forms a homo-nonameric ring structure that serves as a docking platform for flagellar export chaperones in complex with their cognate filament-type substrates. Interactions of the flexible linker of FlhA (FlhAL) with its nearest FlhAC subunit in the ring allow the chaperones to bind to FlhAC to facilitate filament-type protein export, but it remains unclear how it occurs. Here, we report that FlhAL acts as a switch that brings the order to flagellar assembly. The crystal structure of FlhAC(E351A/D356A) showed that Trp-354 in FlhAL bound to the chaperone-binding site of its neighboring subunit. We propose that FlhAL binds to the chaperon-binding site of FlhAC to suppress the interaction between FlhAC and the chaperones until hook assembly is completed.

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