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Sequential unfolding of beta helical protein by single-molecule atomic force microscopy.

Alsteens D, Martinez N, Jamin M, Jacob-Dubuisson F - PLoS ONE (2013)

Bottom Line: In particular, a mechanically resistant subdomain conserved among TpsA proteins and critical for secretion was identified.Hierarchical unfolding of the βhelix in response to a mechanical stress may maintain β-helical portions that can serve as templates for regaining the native structure after stress.The mechanical properties uncovered here might apply to many proteins with β-helical or related folds, both in prokaryotes and in eukaryotes, and play key roles in their structural integrity and functions.

View Article: PubMed Central - PubMed

Affiliation: Université catholique de Louvain, Institute of Condensed Matter and Nanosciences, Louvain-la-Neuve, Belgium. david.alsteens@uclouvain.be

ABSTRACT
The parallel βhelix is a common fold among extracellular proteins, however its mechanical properties remain unexplored. In Gram-negative bacteria, extracellular proteins of diverse functions of the large 'TpsA' family all fold into long βhelices. Here, single-molecule atomic force microscopy and steered molecular dynamics simulations were combined to investigate the mechanical properties of a prototypic TpsA protein, FHA, the major adhesin of Bordetella pertussis. Strong extension forces were required to fully unfold this highly repetitive protein, and unfolding occurred along a stepwise, hierarchical process. Our analyses showed that the extremities of the βhelix unfold early, while central regions of the helix are more resistant to mechanical unfolding. In particular, a mechanically resistant subdomain conserved among TpsA proteins and critical for secretion was identified. This nucleus harbors structural elements packed against the βhelix that might contribute to stabilizing the N-terminal region of FHA. Hierarchical unfolding of the βhelix in response to a mechanical stress may maintain β-helical portions that can serve as templates for regaining the native structure after stress. The mechanical properties uncovered here might apply to many proteins with β-helical or related folds, both in prokaryotes and in eukaryotes, and play key roles in their structural integrity and functions.

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Related in: MedlinePlus

Schematic representation of FHA and model proteins used in this work.(A) Model of FHA. The dotted vertical lines at the right side of the protein model indicate the approximate lengths of Fha60 and Fha30, which both have the same N-terminus as full-length FHA. The TPS domain is shown in blue, the R1 region is shown in red, and the B1, R2 and B2 domains are shown in green, magenta and cyan, respectively. The X-ray structure of Fha30 is known, while the R1 and R2 regions were built by using the models reported in [18], and the B1 and B2 regions were built by molecular modeling using the I-TASSER web server [19]. (B) Conserved regions of Fha30. On the basis of multiple sequence alignments, four regions of different conservation rates were identified in the TPS domain. The most conserved subdomains C1 and C2 are shown in dark blue, and the less conserved subdomains LC1 and LC2 are shown in light blue. The first R1 coils are in red. (C) Structural organization of Fha30. The N-terminal cap and the six successive coils (Coil_A to Coil_F) of the TPS domain are shown in magenta, blue, cyan, green, yellow, orange and brown, respectively. The three R1 coils present in the crystal structure of Fha30 are shown in dark red, red and pink, and the three extra-helical elements are shown in grey. Elements II and III assemble together to form a four-stranded βsheet that packs against the β-helical core formed by coils_A to _F.
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pone-0073572-g001: Schematic representation of FHA and model proteins used in this work.(A) Model of FHA. The dotted vertical lines at the right side of the protein model indicate the approximate lengths of Fha60 and Fha30, which both have the same N-terminus as full-length FHA. The TPS domain is shown in blue, the R1 region is shown in red, and the B1, R2 and B2 domains are shown in green, magenta and cyan, respectively. The X-ray structure of Fha30 is known, while the R1 and R2 regions were built by using the models reported in [18], and the B1 and B2 regions were built by molecular modeling using the I-TASSER web server [19]. (B) Conserved regions of Fha30. On the basis of multiple sequence alignments, four regions of different conservation rates were identified in the TPS domain. The most conserved subdomains C1 and C2 are shown in dark blue, and the less conserved subdomains LC1 and LC2 are shown in light blue. The first R1 coils are in red. (C) Structural organization of Fha30. The N-terminal cap and the six successive coils (Coil_A to Coil_F) of the TPS domain are shown in magenta, blue, cyan, green, yellow, orange and brown, respectively. The three R1 coils present in the crystal structure of Fha30 are shown in dark red, red and pink, and the three extra-helical elements are shown in grey. Elements II and III assemble together to form a four-stranded βsheet that packs against the β-helical core formed by coils_A to _F.

Mentions: The first βhelix to have its structure determined was pectate lyase C (PelC) [3], a relatively small enzyme that contains seven complete βcoils. Since then, several other β?helical proteins have been characterized (e.g. [9]–[14]), which belong notably to two families of secreted bacterial proteins called TpsA proteins and autotransporters [6], [15]. Both families include proteins of several thousand residues predicted to form elongated βhelices. Filamentous haemagglutinin (FHA), the major adhesin of the whooping cough agent Bordetella pertussis[16], is a model TpsA protein. This 230-kDa protein appears by electron microscopy as a rigid, 40 nm-long rod [17]. It is mostly composed of approximately 80 imperfect sequence repeats of 19 residues or more in tandem that form the coils of an elongated βhelix, followed by a predicted globular domain at the C terminus [18] (Fig. 1A). FHA is secreted by the Two-Partner secretion (TPS) pathway [15] and is translocated across the outer membrane by its specific TpsB transporter, called FhaC [20]. Following translocation some FHA is found associated with the cell surface, consistent with its adhesive properties, although some of it is also found into the milieu, presumably exerting immunomodulatory effects [16]. FHA harbours a 250-residue-long N-terminal ‘TPS domain’, which is essential for secretion and conserved among TpsA proteins [12], [13], [21]. The structure of Fha30, a 30-kDa N-terminal fragment that represents the smallest secretion-competent FHA derivative has been determined [12] (Fig. 1B). Fha30 comprises the TPS domain followed by the first three regular ‘R1’ repeats of the central region of FHA, named R1A to R1C (Fig. 1C). The TPS domain forms a right-handed βhelix comprising six irregular coils called A to F that is capped by three β strands at the N terminus. The TPS domain also comprises three extra-helical elements, i.e. a β hairpin between coils_A and _B (extra-helix element I), another βhairpin within coil_D (extra-helix element II) and a βhairpin followed by a loop in coil_F (extra-helix element III). The βhairpins of the extra-helical elements II and III form a βsheet that packs against the βhelix (Fig. 1C). Based on sequence alignments of a number of TpsA proteins, the TPS domain has been subdivided into successive less conserved (LC) and conserved (C) regions called LC1, C1, LC2 and C2, respectively (Fig. 1B) [12]–[14], [21]. In this work, we used a combination of single-molecule AFM [22]–[24] and steered molecular dynamics (SMD) simulations to characterize the molecular elasticity of model FHA proteins. We show that FHA unfolds sequentially when subjected to force and that the TPS domain contains a stable substructure.


Sequential unfolding of beta helical protein by single-molecule atomic force microscopy.

Alsteens D, Martinez N, Jamin M, Jacob-Dubuisson F - PLoS ONE (2013)

Schematic representation of FHA and model proteins used in this work.(A) Model of FHA. The dotted vertical lines at the right side of the protein model indicate the approximate lengths of Fha60 and Fha30, which both have the same N-terminus as full-length FHA. The TPS domain is shown in blue, the R1 region is shown in red, and the B1, R2 and B2 domains are shown in green, magenta and cyan, respectively. The X-ray structure of Fha30 is known, while the R1 and R2 regions were built by using the models reported in [18], and the B1 and B2 regions were built by molecular modeling using the I-TASSER web server [19]. (B) Conserved regions of Fha30. On the basis of multiple sequence alignments, four regions of different conservation rates were identified in the TPS domain. The most conserved subdomains C1 and C2 are shown in dark blue, and the less conserved subdomains LC1 and LC2 are shown in light blue. The first R1 coils are in red. (C) Structural organization of Fha30. The N-terminal cap and the six successive coils (Coil_A to Coil_F) of the TPS domain are shown in magenta, blue, cyan, green, yellow, orange and brown, respectively. The three R1 coils present in the crystal structure of Fha30 are shown in dark red, red and pink, and the three extra-helical elements are shown in grey. Elements II and III assemble together to form a four-stranded βsheet that packs against the β-helical core formed by coils_A to _F.
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Related In: Results  -  Collection

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pone-0073572-g001: Schematic representation of FHA and model proteins used in this work.(A) Model of FHA. The dotted vertical lines at the right side of the protein model indicate the approximate lengths of Fha60 and Fha30, which both have the same N-terminus as full-length FHA. The TPS domain is shown in blue, the R1 region is shown in red, and the B1, R2 and B2 domains are shown in green, magenta and cyan, respectively. The X-ray structure of Fha30 is known, while the R1 and R2 regions were built by using the models reported in [18], and the B1 and B2 regions were built by molecular modeling using the I-TASSER web server [19]. (B) Conserved regions of Fha30. On the basis of multiple sequence alignments, four regions of different conservation rates were identified in the TPS domain. The most conserved subdomains C1 and C2 are shown in dark blue, and the less conserved subdomains LC1 and LC2 are shown in light blue. The first R1 coils are in red. (C) Structural organization of Fha30. The N-terminal cap and the six successive coils (Coil_A to Coil_F) of the TPS domain are shown in magenta, blue, cyan, green, yellow, orange and brown, respectively. The three R1 coils present in the crystal structure of Fha30 are shown in dark red, red and pink, and the three extra-helical elements are shown in grey. Elements II and III assemble together to form a four-stranded βsheet that packs against the β-helical core formed by coils_A to _F.
Mentions: The first βhelix to have its structure determined was pectate lyase C (PelC) [3], a relatively small enzyme that contains seven complete βcoils. Since then, several other β?helical proteins have been characterized (e.g. [9]–[14]), which belong notably to two families of secreted bacterial proteins called TpsA proteins and autotransporters [6], [15]. Both families include proteins of several thousand residues predicted to form elongated βhelices. Filamentous haemagglutinin (FHA), the major adhesin of the whooping cough agent Bordetella pertussis[16], is a model TpsA protein. This 230-kDa protein appears by electron microscopy as a rigid, 40 nm-long rod [17]. It is mostly composed of approximately 80 imperfect sequence repeats of 19 residues or more in tandem that form the coils of an elongated βhelix, followed by a predicted globular domain at the C terminus [18] (Fig. 1A). FHA is secreted by the Two-Partner secretion (TPS) pathway [15] and is translocated across the outer membrane by its specific TpsB transporter, called FhaC [20]. Following translocation some FHA is found associated with the cell surface, consistent with its adhesive properties, although some of it is also found into the milieu, presumably exerting immunomodulatory effects [16]. FHA harbours a 250-residue-long N-terminal ‘TPS domain’, which is essential for secretion and conserved among TpsA proteins [12], [13], [21]. The structure of Fha30, a 30-kDa N-terminal fragment that represents the smallest secretion-competent FHA derivative has been determined [12] (Fig. 1B). Fha30 comprises the TPS domain followed by the first three regular ‘R1’ repeats of the central region of FHA, named R1A to R1C (Fig. 1C). The TPS domain forms a right-handed βhelix comprising six irregular coils called A to F that is capped by three β strands at the N terminus. The TPS domain also comprises three extra-helical elements, i.e. a β hairpin between coils_A and _B (extra-helix element I), another βhairpin within coil_D (extra-helix element II) and a βhairpin followed by a loop in coil_F (extra-helix element III). The βhairpins of the extra-helical elements II and III form a βsheet that packs against the βhelix (Fig. 1C). Based on sequence alignments of a number of TpsA proteins, the TPS domain has been subdivided into successive less conserved (LC) and conserved (C) regions called LC1, C1, LC2 and C2, respectively (Fig. 1B) [12]–[14], [21]. In this work, we used a combination of single-molecule AFM [22]–[24] and steered molecular dynamics (SMD) simulations to characterize the molecular elasticity of model FHA proteins. We show that FHA unfolds sequentially when subjected to force and that the TPS domain contains a stable substructure.

Bottom Line: In particular, a mechanically resistant subdomain conserved among TpsA proteins and critical for secretion was identified.Hierarchical unfolding of the βhelix in response to a mechanical stress may maintain β-helical portions that can serve as templates for regaining the native structure after stress.The mechanical properties uncovered here might apply to many proteins with β-helical or related folds, both in prokaryotes and in eukaryotes, and play key roles in their structural integrity and functions.

View Article: PubMed Central - PubMed

Affiliation: Université catholique de Louvain, Institute of Condensed Matter and Nanosciences, Louvain-la-Neuve, Belgium. david.alsteens@uclouvain.be

ABSTRACT
The parallel βhelix is a common fold among extracellular proteins, however its mechanical properties remain unexplored. In Gram-negative bacteria, extracellular proteins of diverse functions of the large 'TpsA' family all fold into long βhelices. Here, single-molecule atomic force microscopy and steered molecular dynamics simulations were combined to investigate the mechanical properties of a prototypic TpsA protein, FHA, the major adhesin of Bordetella pertussis. Strong extension forces were required to fully unfold this highly repetitive protein, and unfolding occurred along a stepwise, hierarchical process. Our analyses showed that the extremities of the βhelix unfold early, while central regions of the helix are more resistant to mechanical unfolding. In particular, a mechanically resistant subdomain conserved among TpsA proteins and critical for secretion was identified. This nucleus harbors structural elements packed against the βhelix that might contribute to stabilizing the N-terminal region of FHA. Hierarchical unfolding of the βhelix in response to a mechanical stress may maintain β-helical portions that can serve as templates for regaining the native structure after stress. The mechanical properties uncovered here might apply to many proteins with β-helical or related folds, both in prokaryotes and in eukaryotes, and play key roles in their structural integrity and functions.

Show MeSH
Related in: MedlinePlus