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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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Scheme for post-translational transit complex targeting to the thylakoid membrane.(a) Schematic representation of cpSRP43 interactions with linear motifs in post-translational targeting. Flexibility in absence of the binding partners between ankyrin repeats and CD2, and between CD2 and CD3 is indicated with black arrows. (b) Scheme for transit complex interactions at the thylakoid membrane. CpSRP54 establishes the connection to the SRP receptor cpFtsY via targeting complex formation, whereas A3CT tethers the transit complex to the Alb3 insertase within the membrane.
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f5: Scheme for post-translational transit complex targeting to the thylakoid membrane.(a) Schematic representation of cpSRP43 interactions with linear motifs in post-translational targeting. Flexibility in absence of the binding partners between ankyrin repeats and CD2, and between CD2 and CD3 is indicated with black arrows. (b) Scheme for transit complex interactions at the thylakoid membrane. CpSRP54 establishes the connection to the SRP receptor cpFtsY via targeting complex formation, whereas A3CT tethers the transit complex to the Alb3 insertase within the membrane.

Mentions: We now complete the structural portfolio of cpSRP43 interactions by showing that cpSRP43 CD3 specifically interacts with a linear motif in the C-terminal tail of Alb3. We delineate the importance of the domain interfaces in cpSRP43 for recruitment of the transit complex to the thylakoid membrane (Fig. 5a). CD3 recognizes the positively charged motif IV with a central arginine being accommodated in a modified cage with respect to classical chromodomains. The same principle applies to the cpSRP43–cpSRP54 interaction, which is therefore the key-feature of the cpSRP43 chromodomains. Selectivity for CD2 and CD3 is achieved by adaptation of the flanking residues in the interacting linear motifs. CpSRP54 binding to CD2 involves two consecutive arginine residues in the C-terminal tail that are accommodated in a twinned cage and two consecutive proline residues that form a tight β-turn25. Alb3 binding to CD3 is weaker due to distortion of the β-completion and removal of the second cage. Specificity arises as the prolines of the cpSRP54 tail do not fit in CD3 and a serine in the Alb3 tail cannot be accommodated in CD2. In addition, discrimination between motifs II and IV in Alb3 is supported by the read-out of positive charges in the flanking regions of motif IV. Therefore, although the recognized linear motifs are highly similar, each of them contains unique features for distinct recognition. Likewise, preceding domains of cpSRP43 contribute to the ligand read-out in CD2 and CD3: the Ank4 repeat to cpSRP54 and CD2 to Alb3 recognition, respectively. The mode of interface arrangements is also similar and includes the negative dipole of the terminal α helices of the preceding modules that bind respective arginines of the ligands. CpSRP43 contains a third chromodomain at its N terminus (CD1), which shows high sequence and structure conservation2425. However, CD1 has so far not been implicated in interactions with components of LHCP biogenesis. In contrast to CD2 and CD3, the modified cage of CD2 and CD3 is not present in CD1 and a preceding folded domain is missing, which apparently prevents cpSRP54 and Alb3 binding and a ligand, if any, remains to be identified. Interestingly, although A3CT IV (KD 15 μM) contributes most to the overall affinity of A3CT (KD 5 μM) for cpSRP43, additional low affinity interactions involving A3CT-II increase the avidity of the interaction. Previously, fluorescence complementation assays with protoplasts indicated a stronger contribution of motif II (than motif IV) to the interaction with cpSRP43 and an additional binding site in transmembrane domain five of Alb3 (TMD5)40. A recent study in planta described that an Alb3 truncation (including motif IV) results only in a slight reduction in LHCP accumulation compared with wild-type plants when grown under low light conditions41. Our structural and biochemical study focused on the interaction between cpSRP43 CD2CD3 and the Alb3 tail, but additional binding sites in full-length Alb3 may contribute to the interaction in vivo. The presence of multiple linear motifs in the long Alb3 tail suggests that fly-casting contributes to the interaction with cpSRP43.


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)

Scheme for post-translational transit complex targeting to the thylakoid membrane.(a) Schematic representation of cpSRP43 interactions with linear motifs in post-translational targeting. Flexibility in absence of the binding partners between ankyrin repeats and CD2, and between CD2 and CD3 is indicated with black arrows. (b) Scheme for transit complex interactions at the thylakoid membrane. CpSRP54 establishes the connection to the SRP receptor cpFtsY via targeting complex formation, whereas A3CT tethers the transit complex to the Alb3 insertase within the membrane.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4660199&req=5

f5: Scheme for post-translational transit complex targeting to the thylakoid membrane.(a) Schematic representation of cpSRP43 interactions with linear motifs in post-translational targeting. Flexibility in absence of the binding partners between ankyrin repeats and CD2, and between CD2 and CD3 is indicated with black arrows. (b) Scheme for transit complex interactions at the thylakoid membrane. CpSRP54 establishes the connection to the SRP receptor cpFtsY via targeting complex formation, whereas A3CT tethers the transit complex to the Alb3 insertase within the membrane.
Mentions: We now complete the structural portfolio of cpSRP43 interactions by showing that cpSRP43 CD3 specifically interacts with a linear motif in the C-terminal tail of Alb3. We delineate the importance of the domain interfaces in cpSRP43 for recruitment of the transit complex to the thylakoid membrane (Fig. 5a). CD3 recognizes the positively charged motif IV with a central arginine being accommodated in a modified cage with respect to classical chromodomains. The same principle applies to the cpSRP43–cpSRP54 interaction, which is therefore the key-feature of the cpSRP43 chromodomains. Selectivity for CD2 and CD3 is achieved by adaptation of the flanking residues in the interacting linear motifs. CpSRP54 binding to CD2 involves two consecutive arginine residues in the C-terminal tail that are accommodated in a twinned cage and two consecutive proline residues that form a tight β-turn25. Alb3 binding to CD3 is weaker due to distortion of the β-completion and removal of the second cage. Specificity arises as the prolines of the cpSRP54 tail do not fit in CD3 and a serine in the Alb3 tail cannot be accommodated in CD2. In addition, discrimination between motifs II and IV in Alb3 is supported by the read-out of positive charges in the flanking regions of motif IV. Therefore, although the recognized linear motifs are highly similar, each of them contains unique features for distinct recognition. Likewise, preceding domains of cpSRP43 contribute to the ligand read-out in CD2 and CD3: the Ank4 repeat to cpSRP54 and CD2 to Alb3 recognition, respectively. The mode of interface arrangements is also similar and includes the negative dipole of the terminal α helices of the preceding modules that bind respective arginines of the ligands. CpSRP43 contains a third chromodomain at its N terminus (CD1), which shows high sequence and structure conservation2425. However, CD1 has so far not been implicated in interactions with components of LHCP biogenesis. In contrast to CD2 and CD3, the modified cage of CD2 and CD3 is not present in CD1 and a preceding folded domain is missing, which apparently prevents cpSRP54 and Alb3 binding and a ligand, if any, remains to be identified. Interestingly, although A3CT IV (KD 15 μM) contributes most to the overall affinity of A3CT (KD 5 μM) for cpSRP43, additional low affinity interactions involving A3CT-II increase the avidity of the interaction. Previously, fluorescence complementation assays with protoplasts indicated a stronger contribution of motif II (than motif IV) to the interaction with cpSRP43 and an additional binding site in transmembrane domain five of Alb3 (TMD5)40. A recent study in planta described that an Alb3 truncation (including motif IV) results only in a slight reduction in LHCP accumulation compared with wild-type plants when grown under low light conditions41. Our structural and biochemical study focused on the interaction between cpSRP43 CD2CD3 and the Alb3 tail, but additional binding sites in full-length Alb3 may contribute to the interaction in vivo. The presence of multiple linear motifs in the long Alb3 tail suggests that fly-casting contributes to the interaction with cpSRP43.

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
Related in: MedlinePlus