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Structural basis for cpSRP43 chromodomain selectivity and dynamics in Alb3 insertase interaction.

Horn A, Hennig J, Ahmed YL, Stier G, Wild K, Sattler M, Sinning I - Nat Commun (2015)

Bottom Line: Canonical membrane protein biogenesis requires co-translational delivery of ribosome-associated proteins to the Sec translocase and depends on the signal recognition particle (SRP) and its receptor (SR).Negative cooperativity in ligand binding can be explained by dynamics in the chromodomain interface.Our study provides a model for membrane recruitment of the transit complex and may serve as a prototype for a functional gain by the tandem arrangement of chromodomains.

View Article: PubMed Central - PubMed

Affiliation: Heidelberg University Biochemistry Center (BZH), INF 328, Heidelberg D-69120, Germany.

ABSTRACT
Canonical membrane protein biogenesis requires co-translational delivery of ribosome-associated proteins to the Sec translocase and depends on the signal recognition particle (SRP) and its receptor (SR). In contrast, high-throughput delivery of abundant light-harvesting chlorophyll a,b-binding proteins (LHCPs) in chloroplasts to the Alb3 insertase occurs post-translationally via a soluble transit complex including the cpSRP43/cpSRP54 heterodimer (cpSRP). Here we describe the molecular mechanisms of tethering cpSRP to the Alb3 insertase by specific interaction of cpSRP43 chromodomain 3 with a linear motif in the Alb3 C-terminal tail. Combining NMR spectroscopy, X-ray crystallography and biochemical analyses, we dissect the structural basis for selectivity of chromodomains 2 and 3 for their respective ligands cpSRP54 and Alb3, respectively. Negative cooperativity in ligand binding can be explained by dynamics in the chromodomain interface. Our study provides a model for membrane recruitment of the transit complex and may serve as a prototype for a functional gain by the tandem arrangement of chromodomains.

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Structural basis for negative cooperativity of ligand binding to CD2 and CD3.(a) Close-up view on the CD2–CD3–A3CT IV interface. Tyr319 of CD3 forms the hub of the interface. Relevant salt bridges of A3CT IV to CD2 are indicated. Arg460 is fixed on the negative helix dipole of the CD2 C-terminal helix α1. (b) Conformational changes in the CD2–CD3 interface. Top: CD2 in the cpSRP43ΔCD3–RRKRp complex. Gly316 is integral to helix α1. Bottom: CD2CD3–IV complex structure. Gly316 serves as a hinge point (indicated as sphere) and residues 316–319 form strand β2a in the CD2–CD3 interface. (c) Close-up view of NMR spectra around Gly316. In the CD3 constructs (left), Gly316 is the N-terminal residue and is located at a position common for N-terminal glycines. It shifts on A3CT titration and even more if A3CT IV is connected via a GS-linker (CD3—IV). The peak position of Gly316 changes in the CD2CD3 construct (cyan), indicating a conformational change. On binding of RRKRp (right, magenta) the lineshape improves and the peak shifts considerably indicating stabilization likely due to helix formation. Gly316 of RRKRp-preloaded CD2CD3 shifts back on addition of A3CT (cyan), according to its hinge function and the formation of strand β2a.
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f4: Structural basis for negative cooperativity of ligand binding to CD2 and CD3.(a) Close-up view on the CD2–CD3–A3CT IV interface. Tyr319 of CD3 forms the hub of the interface. Relevant salt bridges of A3CT IV to CD2 are indicated. Arg460 is fixed on the negative helix dipole of the CD2 C-terminal helix α1. (b) Conformational changes in the CD2–CD3 interface. Top: CD2 in the cpSRP43ΔCD3–RRKRp complex. Gly316 is integral to helix α1. Bottom: CD2CD3–IV complex structure. Gly316 serves as a hinge point (indicated as sphere) and residues 316–319 form strand β2a in the CD2–CD3 interface. (c) Close-up view of NMR spectra around Gly316. In the CD3 constructs (left), Gly316 is the N-terminal residue and is located at a position common for N-terminal glycines. It shifts on A3CT titration and even more if A3CT IV is connected via a GS-linker (CD3—IV). The peak position of Gly316 changes in the CD2CD3 construct (cyan), indicating a conformational change. On binding of RRKRp (right, magenta) the lineshape improves and the peak shifts considerably indicating stabilization likely due to helix formation. Gly316 of RRKRp-preloaded CD2CD3 shifts back on addition of A3CT (cyan), according to its hinge function and the formation of strand β2a.

Mentions: From the cpSRP43ΔCD3–RRKRp complex it was known that CD2 recognizes the extended 531-PPGTARRKR sequence of cpSRP54 and that the residues flanking the arginine (0) and especially the arginine at the last position (+2) are important for binding25. Our biochemical and structural data for the CD2CD3–IV complex show that also here the extended 454-KRSKRKR sequence of Alb3 is specifically recognized. To understand how the two chromodomains discriminate between similar linear motifs from cpSRP54 and Alb3, we mutated residues from the −7 to the +2 position in A3CT IV to alanine and determined the KD values of single and double point mutants by ITC (Supplementary Table 1a). Residues involved in salt bridges (Arg455, Lys457, Arg458) contribute the most to the binding, which is typical for an entropically unfavourable interaction and reflects the ordering of A3CT IV by formation of strand β1′ during binding. Mutation of the central Lys457 and Arg458 together decreases the dissociation constant by about ninefold (KD of 44 μM) compared with the wild-type interaction. This highlights the importance of Arg458 (0), which is recognized within the modified cage, as the key residue in A3CT IV. Arg455 (−3) forms a salt bridge with Asp273 in CD2 (Fig. 4a) and replacement by alanine results in a sixfold reduction in binding affinity, while the mutation of Ser456 (−2) had only a minor effect (threefold reduction), probably as the small side chain of alanine still fits into place. In general, for sterical reasons chromodomains need an alanine at the −2 position of the ligand, which holds true for the cpSRP54 tail binding to CD2 and for histone tails binding to canonical chromodomains38 (Fig. 3b). This restriction is, however, not valid for the CD3–A3CT IV interaction due to the ‘super-twist' of strand β1′, which creates extra space for a serine residue. The importance of the −2 position for binding specificity is underlined by the recent finding that cpSRP54 tails of green algae of the chlorophyte division have a valine at this position, which specifically inhibits binding to cpSRP43 (ref. 39).


Structural basis for cpSRP43 chromodomain selectivity and dynamics in Alb3 insertase interaction.

Horn A, Hennig J, Ahmed YL, Stier G, Wild K, Sattler M, Sinning I - Nat Commun (2015)

Structural basis for negative cooperativity of ligand binding to CD2 and CD3.(a) Close-up view on the CD2–CD3–A3CT IV interface. Tyr319 of CD3 forms the hub of the interface. Relevant salt bridges of A3CT IV to CD2 are indicated. Arg460 is fixed on the negative helix dipole of the CD2 C-terminal helix α1. (b) Conformational changes in the CD2–CD3 interface. Top: CD2 in the cpSRP43ΔCD3–RRKRp complex. Gly316 is integral to helix α1. Bottom: CD2CD3–IV complex structure. Gly316 serves as a hinge point (indicated as sphere) and residues 316–319 form strand β2a in the CD2–CD3 interface. (c) Close-up view of NMR spectra around Gly316. In the CD3 constructs (left), Gly316 is the N-terminal residue and is located at a position common for N-terminal glycines. It shifts on A3CT titration and even more if A3CT IV is connected via a GS-linker (CD3—IV). The peak position of Gly316 changes in the CD2CD3 construct (cyan), indicating a conformational change. On binding of RRKRp (right, magenta) the lineshape improves and the peak shifts considerably indicating stabilization likely due to helix formation. Gly316 of RRKRp-preloaded CD2CD3 shifts back on addition of A3CT (cyan), according to its hinge function and the formation of strand β2a.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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f4: Structural basis for negative cooperativity of ligand binding to CD2 and CD3.(a) Close-up view on the CD2–CD3–A3CT IV interface. Tyr319 of CD3 forms the hub of the interface. Relevant salt bridges of A3CT IV to CD2 are indicated. Arg460 is fixed on the negative helix dipole of the CD2 C-terminal helix α1. (b) Conformational changes in the CD2–CD3 interface. Top: CD2 in the cpSRP43ΔCD3–RRKRp complex. Gly316 is integral to helix α1. Bottom: CD2CD3–IV complex structure. Gly316 serves as a hinge point (indicated as sphere) and residues 316–319 form strand β2a in the CD2–CD3 interface. (c) Close-up view of NMR spectra around Gly316. In the CD3 constructs (left), Gly316 is the N-terminal residue and is located at a position common for N-terminal glycines. It shifts on A3CT titration and even more if A3CT IV is connected via a GS-linker (CD3—IV). The peak position of Gly316 changes in the CD2CD3 construct (cyan), indicating a conformational change. On binding of RRKRp (right, magenta) the lineshape improves and the peak shifts considerably indicating stabilization likely due to helix formation. Gly316 of RRKRp-preloaded CD2CD3 shifts back on addition of A3CT (cyan), according to its hinge function and the formation of strand β2a.
Mentions: From the cpSRP43ΔCD3–RRKRp complex it was known that CD2 recognizes the extended 531-PPGTARRKR sequence of cpSRP54 and that the residues flanking the arginine (0) and especially the arginine at the last position (+2) are important for binding25. Our biochemical and structural data for the CD2CD3–IV complex show that also here the extended 454-KRSKRKR sequence of Alb3 is specifically recognized. To understand how the two chromodomains discriminate between similar linear motifs from cpSRP54 and Alb3, we mutated residues from the −7 to the +2 position in A3CT IV to alanine and determined the KD values of single and double point mutants by ITC (Supplementary Table 1a). Residues involved in salt bridges (Arg455, Lys457, Arg458) contribute the most to the binding, which is typical for an entropically unfavourable interaction and reflects the ordering of A3CT IV by formation of strand β1′ during binding. Mutation of the central Lys457 and Arg458 together decreases the dissociation constant by about ninefold (KD of 44 μM) compared with the wild-type interaction. This highlights the importance of Arg458 (0), which is recognized within the modified cage, as the key residue in A3CT IV. Arg455 (−3) forms a salt bridge with Asp273 in CD2 (Fig. 4a) and replacement by alanine results in a sixfold reduction in binding affinity, while the mutation of Ser456 (−2) had only a minor effect (threefold reduction), probably as the small side chain of alanine still fits into place. In general, for sterical reasons chromodomains need an alanine at the −2 position of the ligand, which holds true for the cpSRP54 tail binding to CD2 and for histone tails binding to canonical chromodomains38 (Fig. 3b). This restriction is, however, not valid for the CD3–A3CT IV interaction due to the ‘super-twist' of strand β1′, which creates extra space for a serine residue. The importance of the −2 position for binding specificity is underlined by the recent finding that cpSRP54 tails of green algae of the chlorophyte division have a valine at this position, which specifically inhibits binding to cpSRP43 (ref. 39).

Bottom Line: Canonical membrane protein biogenesis requires co-translational delivery of ribosome-associated proteins to the Sec translocase and depends on the signal recognition particle (SRP) and its receptor (SR).Negative cooperativity in ligand binding can be explained by dynamics in the chromodomain interface.Our study provides a model for membrane recruitment of the transit complex and may serve as a prototype for a functional gain by the tandem arrangement of chromodomains.

View Article: PubMed Central - PubMed

Affiliation: Heidelberg University Biochemistry Center (BZH), INF 328, Heidelberg D-69120, Germany.

ABSTRACT
Canonical membrane protein biogenesis requires co-translational delivery of ribosome-associated proteins to the Sec translocase and depends on the signal recognition particle (SRP) and its receptor (SR). In contrast, high-throughput delivery of abundant light-harvesting chlorophyll a,b-binding proteins (LHCPs) in chloroplasts to the Alb3 insertase occurs post-translationally via a soluble transit complex including the cpSRP43/cpSRP54 heterodimer (cpSRP). Here we describe the molecular mechanisms of tethering cpSRP to the Alb3 insertase by specific interaction of cpSRP43 chromodomain 3 with a linear motif in the Alb3 C-terminal tail. Combining NMR spectroscopy, X-ray crystallography and biochemical analyses, we dissect the structural basis for selectivity of chromodomains 2 and 3 for their respective ligands cpSRP54 and Alb3, respectively. Negative cooperativity in ligand binding can be explained by dynamics in the chromodomain interface. Our study provides a model for membrane recruitment of the transit complex and may serve as a prototype for a functional gain by the tandem arrangement of chromodomains.

Show MeSH