Solution Structures of Two CCHC Zinc Fingers from the FOG Family Protein U-Shaped that Mediate Protein–Protein Interactions

Liew, Chu Kong; Kowalski, Kasper; Fox, Archa H.; Newton, Anthea L.; Sharpe, B.K.; Crossley, Merlin; Mackay, Joel P.

doi:10.1016/s0969-2126(00)00527-x

Cited by 46 publications

(40 citation statements)

References 44 publications

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“…NOESY cross-peak volumes were estimated using the Guassian fit mode in Sparky and ϕ angle constraints derived from a HNHA (51). A total of 100, 20, and 1,000 structures were calculated in iterations 0, 2-7, and 8, respectively.…”

Section: Methodsmentioning

confidence: 99%

Self-assembly of functional, amphipathic amyloid monolayers by the fungal hydrophobin EAS

Macindoe

Kwan

Ren

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

121

111

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The hydrophobin EAS from the fungus Neurospora crassa forms functional amyloid fibrils called rodlets that facilitate spore formation and dispersal. Self-assembly of EAS into fibrillar rodlets occurs spontaneously at hydrophobic:hydrophilic interfaces and the rodlets further associate laterally to form amphipathic monolayers. We have used site-directed mutagenesis and peptide experiments to identify the region of EAS that drives intermolecular association and formation of the cross-β rodlet structure. Transplanting this region into a nonamyloidogenic hydrophobin enables it to form rodlets. We have also determined the structure and dynamics of an EAS variant with reduced rodlet-forming ability. Taken together, these data allow us to pinpoint the conformational changes that take place when hydrophobins self-assemble at an interface and to propose a model for the amphipathic EAS rodlet structure.A myloid fibrils were first identified in association with human diseases, but recent discoveries show that the amyloid ultrastructure also contributes to important functions in normal biology (1, 2). In bacteria, fungi, insects, fish, and mammals, amyloid structures perform a wide variety of roles (3). Functional amyloids in the form of fibrillar rodlets composed of class I hydrophobin proteins are found in filamentous fungi. These hydrophobins are small proteins that are secreted as monomers and self-assemble into rodlets that pack to form amphipathic monolayers at hydrophilic: hydrophobic boundaries, such as the surface of the growth medium (4). These proteins are extremely surface active and lower the surface tension of the aqueous growth medium, allowing hyphae to break through the surface and to produce aerial structures (5, 6). Many of these aerial structures subsequently become coated with amyloid rodlets, creating a hydrophobic layer that serves multiple purposes, including conferring water resistance to spores for easier dispersal in air (7), preventing wetting or collapse of gas transfer channels (8), enhancing adherence to waxy surfaces such as leaves during infection of rice plants by Magnaporthe grisea (9), and mediating evasion of the immune system as is observed in Aspergillus fumigatus infections (10).Hydrophobins are characterized by the presence of eight cysteine residues that form four disulphide bonds, but the hydrophobin family can be further divided into two classes based on the spacing of the conserved cysteine residues and the nature of the amphipathic monolayers that they form (11). Class I, but not class II, hydrophobins form amyloid-like rodlets that are extremely robust and require treatment with strong acid to induce depolymerization. The amphipathic monolayers formed by class II hydrophobins are not fibrillar and can be dissociated by treatment with detergent and alcohol solutions. The soluble, monomeric forms of hydrophobins share a unique β-barrel topology, and all have a relatively large exposed hydrophobic area on the protein monomer surface (4). The diversity in sequence and chain length ...

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Section: Methodsmentioning

confidence: 99%

Self-assembly of functional, amphipathic amyloid monolayers by the fungal hydrophobin EAS

Macindoe

Kwan

Ren

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

121

111

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“…Expression and Purification-USH-F1, USH-F9, and murine GATA-1 NF were expressed and purified as previously described (16,23). Pannier NF (Pannier (165-208)) was subcloned, overexpressed, and purified using the same protocol as that used for the GATA-1 NF, except that the template for PCR of the construct was obtained from a D. melanogaster cDNA library.…”

Section: Methodsmentioning

confidence: 99%

“…ϭ 4202.0 Da). Overexpression of 15 N-labeled C32H was performed using a bacterial fermenter as previously described (16).…”

Section: Methodsmentioning

confidence: 99%

“…The structures of two of these domains (fingers 1 and 9 from D. melanogaster U-shaped) have recently been determined (16) and were found to resemble classical CCHH fingers in structure. The only notable difference is a short extended portion of the polypeptide backbone before the fourth zinc ligand (Fig.…”

mentioning

confidence: 99%

“…1C). The GATA binding surface of one CCHC finger (finger 1 from U-shaped; USH-F1) has been determined (16,17). Two of the residues implicated in GATA binding are located on the extended portion of the backbone, immediately preceding the final cysteine, and mutation of the cysteine to histidine (changing the finger to a classical CCHH ZnF) is sufficient to abolish the GATA-FOG interaction in vivo (18).…”

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confidence: 99%

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Characterization of the Conserved Interaction between GATA and FOG Family Proteins

Kowalski¹,

Liew²,

Matthews³

et al. 2002

Journal of Biological Chemistry

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The N-terminal zinc finger (ZnF) from GATA transcription factors mediates interactions with FOG family proteins. In FOG proteins, the interacting domains are also ZnFs; these domains are related to classical CCHH fingers but have an His 3 Cys substitution at the final zinc-ligating position. Here we demonstrate that different CCHC fingers in the FOG family protein U-shaped contact the N-terminal ZnF of GATA-1 in the same fashion although with different affinities. We also show that these interactions are of moderate affinity, which is interesting given the presumed low concentrations of these proteins in the nucleus. Furthermore, we demonstrate that the variant CCHC topology enhances binding affinity, although the His 3 Cys change is not essential for the formation of a stably folded domain. To ascertain the structural basis for the contribution of the CCHC arrangement, we have determined the structure of a CCHH mutant of finger nine from U-shaped. The structure is very similar overall to the wild-type domain, with subtle differences at the C terminus that result in loss of the interaction in vivo. Taken together, these results suggest that the CCHC zinc binding topology is required for the integrity of GATA-FOG interactions and that weak interactions can play important roles in vivo.

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The Protein-Binding Potential of C2H2 Zinc Finger Domains

Brayer

Kulshreshtha

Segal

2008

Cell Biochem Biophys

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Solution Structures of Two CCHC Zinc Fingers from the FOG Family Protein U-Shaped that Mediate Protein–Protein Interactions

Cited by 46 publications

References 44 publications

Self-assembly of functional, amphipathic amyloid monolayers by the fungal hydrophobin EAS

Self-assembly of functional, amphipathic amyloid monolayers by the fungal hydrophobin EAS

Characterization of the Conserved Interaction between GATA and FOG Family Proteins

The Protein-Binding Potential of C2H2 Zinc Finger Domains

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