Cooperative folding of intrinsically disordered domains drives assembly of a strong elongated protein

Gruszka, Dominika T.; Whelan, Fiona; Farrance, Oliver E.; Fung, Herman K H; Paci, Emanuele; Jeffries, Cy M.; Svergun, Dmitri I.; Baldock, Clair; Baumann, Christoph G.; Brockwell, David J.; Potts, Jennifer R.; Clarke, Jane

doi:10.1038/ncomms8271

Cited by 58 publications

(79 citation statements)

References 70 publications

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“…Because SasG domains have no tryptophans, (un)folding was followed by monitoring intrinsic tyrosine fluorescence. We have shown that ureainduced equilibrium denaturation curves of E-G5 2 monitored by fluorescence coincide with those recorded by ellipticity at 235 nm (7) and domain-specific FRET probes (9), showing that equilibrium unfolding of the two-domain construct is fully cooperative: two-state with concerted disruption of both domains and secondary and tertiary structure with no accumulation of intermediates (Fig. 1C).…”

Section: Resultssupporting

confidence: 57%

“…We had previously shown that disorder-mediated thermodynamic cooperativity allows SasG to adopt long, mechanically strong, rod-like structures (9). Now, we have shown how this disorder coupled with the remarkable stability of the interdomain interface can result in cooperative folding kinetics, with no populated intermediates, across long distances.…”

Section: Resultsmentioning

confidence: 83%

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Disorder drives cooperative folding in a multidomain protein

Gruszka

Mendonca

Paci

et al. 2016

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

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Many human proteins contain intrinsically disordered regions, and disorder in these proteins can be fundamental to their function-for example, facilitating transient but specific binding, promoting allostery, or allowing efficient posttranslational modification. SasG, a multidomain protein implicated in host colonization and biofilm formation in Staphylococcus aureus, provides another example of how disorder can play an important role. Approximately one-half of the domains in the extracellular repetitive region of SasG are intrinsically unfolded in isolation, but these E domains fold in the context of their neighboring folded G5 domains. We have previously shown that the intrinsic disorder of the E domains mediates long-range cooperativity between nonneighboring G5 domains, allowing SasG to form a long, rod-like, mechanically strong structure. Here, we show that the disorder of the E domains coupled with the remarkable stability of the interdomain interface result in cooperative folding kinetics across long distances. Formation of a small structural nucleus at one end of the molecule results in rapid structure formation over a distance of 10 nm, which is likely to be important for the maintenance of the structural integrity of SasG. Moreover, if this normal folding nucleus is disrupted by mutation, the interdomain interface is sufficiently stable to drive the folding of adjacent E and G5 domains along a parallel folding pathway, thus maintaining cooperative folding.IDP | protein folding | parallel pathways | protein engineering | cooperativity I t has been suggested that as much as 20% of the proteome may be intrinsically disordered (1), mainly manifested as intrinsically disordered regions within multidomain proteins, although a few proteins are apparently entirely disordered. Some proteins function as a consequence of disorder: for example, disordered PEVK regions of titin act as an entropic spring (2), whereas in the nuclear pore complex, disordered nucleoporins provide a thick selective barrier controlling nuclear import (3). Disorder can also play other roles: it facilitates posttranslational modification and may promote allostery (4, 5). SasG (Staphylococcus aureus surface protein G) is a cell wall-attached protein from S. aureus that promotes intercellular adhesion during the accumulation phase of biofilm formation via its C-terminal repetitive region (6-8). We previously showed that this part of SasG contains alternating E and G5 domains (Fig. 1A) and that E folds when it is N-terminal of a G5 domain. The disorder of E domains in isolation is essential for formation of a long, stiff, mechanically strong, rod-like structure (9) capable of projecting the N-terminal A domain, which is involved in host colonization (6).Here, we combine biophysical measurements, protein engineering, and simulation to show that the disorder in the E domains of SasG also promotes cooperative folding and unfolding pathways. We find that SasG domains have a highly polarized transition-state (TS) structure, where formation of a sm...

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Section: Resultssupporting

confidence: 57%

Section: Resultsmentioning

confidence: 83%

Disorder drives cooperative folding in a multidomain protein

Gruszka

Mendonca

Paci

et al. 2016

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

Self Cite

View full text Add to dashboard Cite

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“…In addition, 235 HN crosspeaks were identified, which is very close to the expected number (238). The presence of crowded resonances in the center of the 1 H, 15 N TROSY-HSQC spectrum of Phafin2 is consistent with the presence of random coil regions in the protein as deduced from far-UV CD analysis. NMR spectra of Phafin2 at higher temperatures than 308C were also recorded, yielding loss of resonances, and aggregation and precipitation of the protein (data not shown).…”

Section: Nmr Studies Of Phafin2supporting

confidence: 81%

“…Figure 6 shows that the 1 H, 15 N TROSY-HSQC spectrum of Phafin2 yielded a single set of resonances, indicating that the protein was highly homogenous at 308C. In addition, 235 HN crosspeaks were identified, which is very close to the expected number (238).…”

Section: Nmr Studies Of Phafin2mentioning

confidence: 80%

“…In the case of GuHCl-mediated denaturation, the Phafin2 structure was stable at 0.5M of GuHCl concentration, but achieved full denaturation at 3M of the denaturant using both methodologies. Unfolding of small globular proteins occurs through a process that conforms to the two-state mechanism 12 ; however, elongated proteins, such as the Notch ankyrin domain, 13 spectrin, 14 and the bacterial surface protein SasG, 15 among others, exhibit chemical denaturation sigmoidal plots. In this scenario, the population level of intermediate states is insignificant.…”

Section: Denaturant-induced Unfolding Of Phafin2mentioning

confidence: 99%

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Structural, Thermodynamic and Phosphatidylinositol 3-Phosphate Binding Properties of Phafin2

Tang

2017

Biophysical Journal

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Phafin2 is a phosphatidylinositol 3-phosphate (PtdIns(3)P) binding protein involved in the regulation of endosomal cargo trafficking and lysosomal induction of autophagy. Binding of Phafin2 to PtdIns(3)P is mediated by both its PH and FYVE domains. However, there are no studies on the structural basis, conformational stability, and lipid interactions of Phafin2 to better understand how this protein participates in signaling at the surface of endomembrane compartments. Here, we show that human Phafin2 is a moderately elongated monomer of~28 kDa with an intensityaverage hydrodynamic diameter of~7 nm. Circular dichroism (CD) analysis indicates that Phafin2 exhibits an a/b structure and predicts~40% random coil content in the protein. Heteronuclear NMR data indicates that a unique conformation of Phafin2 is present in solution and dispersion of resonances suggests that the protein exhibits random coiled regions, in agreement with the CD data. Phafin2 is stable, displaying a melting temperature of 48.48C. The folding-unfolding curves, obtained using urea-and guanidine hydrochloride-mediated denaturation, indicate that Phafin2 undergoes a two-state native-to-denatured transition. Analysis of these transitions shows that the free energy change for urea-and guanidine hydrochloride-induced Phafin2 denaturation in water is 4 kcal mol 21 . PtdIns(3)P binding to Phafin2 occurs with high affinity, triggering minor conformational changes in the protein. Taken together, these studies represent a platform for establishing the structural basis of Phafin2 molecular interactions and the role of the two potentially redundant PtdIns(3)P-binding domains of the protein in endomembrane compartments.

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Transiently disordered tails accelerate folding of globular proteins

2017

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Numerous biological proteins exhibit intrinsic disorder at their termini, which are associated with multifarious functional roles. Here, we show the surprising result that an increased percentage of terminal short transiently disordered regions with enhanced flexibility (TstDREF) is associated with accelerated folding rates of globular proteins. Evolutionary conservation of predicted disorder at TstDREFs and drastic alteration of folding rates upon point-mutations suggest critical regulatory role(s) of TstDREFs in shaping the folding kinetics. TstDREFs are associated with long-range intramolecular interactions and the percentage of native secondary structural elements physically contacted by TstDREFs exhibit another surprising positive correlation with folding kinetics. These results allow us to infer probable molecular mechanisms behind the TstDREF-mediated regulation of folding kinetics that challenge protein biochemists to assess by direct experimental testing.

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Cooperative folding of intrinsically disordered domains drives assembly of a strong elongated protein

Cited by 58 publications

References 70 publications

Disorder drives cooperative folding in a multidomain protein

Disorder drives cooperative folding in a multidomain protein

Structural, Thermodynamic and Phosphatidylinositol 3-Phosphate Binding Properties of Phafin2

Transiently disordered tails accelerate folding of globular proteins

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