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Structures of actin-like ParM filaments show architecture of plasmid-segregating spindles.

Bharat TA, Murshudov GN, Sachse C, Löwe J - Nature (2015)

Bottom Line: Growing ParMRC spindles push sister plasmids to the cell poles.The ParM filament structure shows strong longitudinal interfaces and weaker lateral interactions.Finally, with whole-cell electron cryotomography, we show that doublets are abundant in bacterial cells containing low-copy-number plasmids with the ParMRC locus, leading to an asynchronous model of R1 plasmid segregation.

View Article: PubMed Central - PubMed

Affiliation: Structural Studies Division, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.

ABSTRACT
Active segregation of Escherichia coli low-copy-number plasmid R1 involves formation of a bipolar spindle made of left-handed double-helical actin-like ParM filaments. ParR links the filaments with centromeric parC plasmid DNA, while facilitating the addition of subunits to ParM filaments. Growing ParMRC spindles push sister plasmids to the cell poles. Here, using modern electron cryomicroscopy methods, we investigate the structures and arrangements of ParM filaments in vitro and in cells, revealing at near-atomic resolution how subunits and filaments come together to produce the simplest known mitotic machinery. To understand the mechanism of dynamic instability, we determine structures of ParM filaments in different nucleotide states. The structure of filaments bound to the ATP analogue AMPPNP is determined at 4.3 Å resolution and refined. The ParM filament structure shows strong longitudinal interfaces and weaker lateral interactions. Also using electron cryomicroscopy, we reconstruct ParM doublets forming antiparallel spindles. Finally, with whole-cell electron cryotomography, we show that doublets are abundant in bacterial cells containing low-copy-number plasmids with the ParMRC locus, leading to an asynchronous model of R1 plasmid segregation.

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ParM filaments are made up of two protofilaments held together by salt bridges, which are perturbed when ParM is bound to ADP(a) The refined atomic model of ParM+AMPPNP filaments shows that the protofilaments are held together laterally by salt bridges. Basic residues at the interface are highlighted in red and acidic residues in orange (see ED Table 2). Within the protofilaments’ longitudinal interfaces, more extensive hydrophobic interactions are observed (see ED Fig. 2). (b) A magnified view of a). The charges of two basic residues at the interface were inverted by mutation for panel c) (K258D, R262D). (c) The resulting protein formed filaments inefficiently. Cryo-EM image showing filaments of ParM(K258D, R262D) assembled with AMPPNP. This experiment was repeated four times. (d) In addition to normal double-helical filaments, some single-helical filaments were observed by image classification and averaging. (e) Fourier shell correlation (FSC) curves for the four cryo-EM structures presented in this study (see ED Table 1). (f) Cryo-EM image of ParM+ADP filaments. High protein concentrations were required to obtain these filaments and monomeric proteins can be seen. This experiment was repeated six times. (g) Comparison of filtered class averages of ParM+ATP and ParM+ADP filaments. Compared to the ATP bound state, the pitch of the ParM+ADP filaments reduced by ~ 3 Å (see Video 2). (h) Cryo-EM reconstruction of ParM+ADP filaments at 11 Å resolution with 5 copies of the ParM+ADP X-ray structure fitted. (i) The same pseudo-atomic fit without the cryo-EM density. (j) A magnified view of the perturbed inter-protofilament interface in the ParM+ADP filaments.
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Figure 2: ParM filaments are made up of two protofilaments held together by salt bridges, which are perturbed when ParM is bound to ADP(a) The refined atomic model of ParM+AMPPNP filaments shows that the protofilaments are held together laterally by salt bridges. Basic residues at the interface are highlighted in red and acidic residues in orange (see ED Table 2). Within the protofilaments’ longitudinal interfaces, more extensive hydrophobic interactions are observed (see ED Fig. 2). (b) A magnified view of a). The charges of two basic residues at the interface were inverted by mutation for panel c) (K258D, R262D). (c) The resulting protein formed filaments inefficiently. Cryo-EM image showing filaments of ParM(K258D, R262D) assembled with AMPPNP. This experiment was repeated four times. (d) In addition to normal double-helical filaments, some single-helical filaments were observed by image classification and averaging. (e) Fourier shell correlation (FSC) curves for the four cryo-EM structures presented in this study (see ED Table 1). (f) Cryo-EM image of ParM+ADP filaments. High protein concentrations were required to obtain these filaments and monomeric proteins can be seen. This experiment was repeated six times. (g) Comparison of filtered class averages of ParM+ATP and ParM+ADP filaments. Compared to the ATP bound state, the pitch of the ParM+ADP filaments reduced by ~ 3 Å (see Video 2). (h) Cryo-EM reconstruction of ParM+ADP filaments at 11 Å resolution with 5 copies of the ParM+ADP X-ray structure fitted. (i) The same pseudo-atomic fit without the cryo-EM density. (j) A magnified view of the perturbed inter-protofilament interface in the ParM+ADP filaments.

Mentions: Surprisingly, the two protofilaments (strands) making up the double-helical ParM filament are held together only by salt bridges (Fig. 2a-b, ED Fig. 2-3 and ED Table 2). The ParM inter-protofilament interface is small (calculated interface area 371 Å2) and does not resemble a canonical protein-protein interface containing a hydrophobic core. To demonstrate the validity of this assessment we mutated two positively charged residues within the inter-protofilament interface to aspartic acids (K258D, R262D) and tested what effect this has on the stability of ParM filaments. Filament formation (with AMPPNP) from the resulting mutant protein ParM (K258D, R262D) was inefficient (ED Fig. 3g). The few filaments that were formed were unstable, and tended to be bent (Fig. 2c, S3h). Reference-free class averaging of these filaments showed that even though the majority of the few observed filaments were double helical like wild-type ParM, some single-helical filaments were also present (Fig. 2d, S3i). These observations indicate that although the interface between protofilaments in ParM is surprisingly small, it is sufficient for double filament assembly since many identical contacts along the filament contribute to the overall binding energy. Different actin-like proteins show very different filament arrangements, from single (crenactin, possibly 11) to parallel double helical (left-handed: ParM, right-handed: actin and non-staggered: MamK 12) and antiparallel, double straight (MreB). We propose that small and simple inter-protofilament contacts could have made it possible to change inter-protofilament arrangements relatively easily during evolution since all these actin-like filaments show similar longitudinal contacts 13.


Structures of actin-like ParM filaments show architecture of plasmid-segregating spindles.

Bharat TA, Murshudov GN, Sachse C, Löwe J - Nature (2015)

ParM filaments are made up of two protofilaments held together by salt bridges, which are perturbed when ParM is bound to ADP(a) The refined atomic model of ParM+AMPPNP filaments shows that the protofilaments are held together laterally by salt bridges. Basic residues at the interface are highlighted in red and acidic residues in orange (see ED Table 2). Within the protofilaments’ longitudinal interfaces, more extensive hydrophobic interactions are observed (see ED Fig. 2). (b) A magnified view of a). The charges of two basic residues at the interface were inverted by mutation for panel c) (K258D, R262D). (c) The resulting protein formed filaments inefficiently. Cryo-EM image showing filaments of ParM(K258D, R262D) assembled with AMPPNP. This experiment was repeated four times. (d) In addition to normal double-helical filaments, some single-helical filaments were observed by image classification and averaging. (e) Fourier shell correlation (FSC) curves for the four cryo-EM structures presented in this study (see ED Table 1). (f) Cryo-EM image of ParM+ADP filaments. High protein concentrations were required to obtain these filaments and monomeric proteins can be seen. This experiment was repeated six times. (g) Comparison of filtered class averages of ParM+ATP and ParM+ADP filaments. Compared to the ATP bound state, the pitch of the ParM+ADP filaments reduced by ~ 3 Å (see Video 2). (h) Cryo-EM reconstruction of ParM+ADP filaments at 11 Å resolution with 5 copies of the ParM+ADP X-ray structure fitted. (i) The same pseudo-atomic fit without the cryo-EM density. (j) A magnified view of the perturbed inter-protofilament interface in the ParM+ADP filaments.
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Related In: Results  -  Collection

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Figure 2: ParM filaments are made up of two protofilaments held together by salt bridges, which are perturbed when ParM is bound to ADP(a) The refined atomic model of ParM+AMPPNP filaments shows that the protofilaments are held together laterally by salt bridges. Basic residues at the interface are highlighted in red and acidic residues in orange (see ED Table 2). Within the protofilaments’ longitudinal interfaces, more extensive hydrophobic interactions are observed (see ED Fig. 2). (b) A magnified view of a). The charges of two basic residues at the interface were inverted by mutation for panel c) (K258D, R262D). (c) The resulting protein formed filaments inefficiently. Cryo-EM image showing filaments of ParM(K258D, R262D) assembled with AMPPNP. This experiment was repeated four times. (d) In addition to normal double-helical filaments, some single-helical filaments were observed by image classification and averaging. (e) Fourier shell correlation (FSC) curves for the four cryo-EM structures presented in this study (see ED Table 1). (f) Cryo-EM image of ParM+ADP filaments. High protein concentrations were required to obtain these filaments and monomeric proteins can be seen. This experiment was repeated six times. (g) Comparison of filtered class averages of ParM+ATP and ParM+ADP filaments. Compared to the ATP bound state, the pitch of the ParM+ADP filaments reduced by ~ 3 Å (see Video 2). (h) Cryo-EM reconstruction of ParM+ADP filaments at 11 Å resolution with 5 copies of the ParM+ADP X-ray structure fitted. (i) The same pseudo-atomic fit without the cryo-EM density. (j) A magnified view of the perturbed inter-protofilament interface in the ParM+ADP filaments.
Mentions: Surprisingly, the two protofilaments (strands) making up the double-helical ParM filament are held together only by salt bridges (Fig. 2a-b, ED Fig. 2-3 and ED Table 2). The ParM inter-protofilament interface is small (calculated interface area 371 Å2) and does not resemble a canonical protein-protein interface containing a hydrophobic core. To demonstrate the validity of this assessment we mutated two positively charged residues within the inter-protofilament interface to aspartic acids (K258D, R262D) and tested what effect this has on the stability of ParM filaments. Filament formation (with AMPPNP) from the resulting mutant protein ParM (K258D, R262D) was inefficient (ED Fig. 3g). The few filaments that were formed were unstable, and tended to be bent (Fig. 2c, S3h). Reference-free class averaging of these filaments showed that even though the majority of the few observed filaments were double helical like wild-type ParM, some single-helical filaments were also present (Fig. 2d, S3i). These observations indicate that although the interface between protofilaments in ParM is surprisingly small, it is sufficient for double filament assembly since many identical contacts along the filament contribute to the overall binding energy. Different actin-like proteins show very different filament arrangements, from single (crenactin, possibly 11) to parallel double helical (left-handed: ParM, right-handed: actin and non-staggered: MamK 12) and antiparallel, double straight (MreB). We propose that small and simple inter-protofilament contacts could have made it possible to change inter-protofilament arrangements relatively easily during evolution since all these actin-like filaments show similar longitudinal contacts 13.

Bottom Line: Growing ParMRC spindles push sister plasmids to the cell poles.The ParM filament structure shows strong longitudinal interfaces and weaker lateral interactions.Finally, with whole-cell electron cryotomography, we show that doublets are abundant in bacterial cells containing low-copy-number plasmids with the ParMRC locus, leading to an asynchronous model of R1 plasmid segregation.

View Article: PubMed Central - PubMed

Affiliation: Structural Studies Division, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.

ABSTRACT
Active segregation of Escherichia coli low-copy-number plasmid R1 involves formation of a bipolar spindle made of left-handed double-helical actin-like ParM filaments. ParR links the filaments with centromeric parC plasmid DNA, while facilitating the addition of subunits to ParM filaments. Growing ParMRC spindles push sister plasmids to the cell poles. Here, using modern electron cryomicroscopy methods, we investigate the structures and arrangements of ParM filaments in vitro and in cells, revealing at near-atomic resolution how subunits and filaments come together to produce the simplest known mitotic machinery. To understand the mechanism of dynamic instability, we determine structures of ParM filaments in different nucleotide states. The structure of filaments bound to the ATP analogue AMPPNP is determined at 4.3 Å resolution and refined. The ParM filament structure shows strong longitudinal interfaces and weaker lateral interactions. Also using electron cryomicroscopy, we reconstruct ParM doublets forming antiparallel spindles. Finally, with whole-cell electron cryotomography, we show that doublets are abundant in bacterial cells containing low-copy-number plasmids with the ParMRC locus, leading to an asynchronous model of R1 plasmid segregation.

Show MeSH
Related in: MedlinePlus