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Structure of a nucleotide-bound Clp1-Pcf11 polyadenylation factor.

Noble CG, Beuth B, Taylor IA - Nucleic Acids Res. (2006)

Bottom Line: The arrangement of the nucleotide binding site is similar to that observed in SIMIBI-class ATPase subunits found in other multisubunit macromolecular complexes.However, despite this similarity, nucleotide hydrolysis does not occur.Moreover, we suggest that this complex represents a stabilized ATP bound form of Clp1 that requires the participation of other non-CFIA processing factors in order to initiate timely ATP hydrolysis during 3' end processing.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Structure, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.

ABSTRACT
Pcf11 and Clp1 are subunits of cleavage factor IA (CFIA), an essential polyadenylation factor in Saccahromyces cerevisiae. We have determined the structure of a ternary complex of Clp1 together with ATP and the Clp1-binding region of Pcf11. Clp1 contains three domains, a small N-terminal beta sandwich domain, a C-terminal domain containing a novel alpha/beta-fold and a central domain that binds ATP. The arrangement of the nucleotide binding site is similar to that observed in SIMIBI-class ATPase subunits found in other multisubunit macromolecular complexes. However, despite this similarity, nucleotide hydrolysis does not occur. The Pcf11 binding site is also located in the central domain where three highly conserved residues in Pcf11 mediate many of the protein-protein interactions. We propose that this conserved Clp1-Pcf11 interaction is responsible for maintaining a tight coupling between the Clp1 nucleotide binding subunit and the other components of the polyadenylation machinery. Moreover, we suggest that this complex represents a stabilized ATP bound form of Clp1 that requires the participation of other non-CFIA processing factors in order to initiate timely ATP hydrolysis during 3' end processing.

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The switch loops and Mg2+ co-ordination. (a) An overlap of the Clp1-ATP (green) and NifH-AlF4 (blue) structures around the P-loop, Switch I and switch II nucleotide binding regions. The backbone representation for each molecule is shown together with a stick representation for the ATP or ADP-AlF4. (b) Interactions of the magnesium ion with the switch loops of Clp1. Stick representations of the P-loop, Switch I and Switch II are shown in light red, yellow and blue, respectively. The adenine base of the ATP is shown in cyan and the magnesium ion is shown as a blue sphere. The water molecule co-ordinated by the magnesium ion and hydrogen bonded by the side-chain of Q164 together with α phosphate oxygen is shown as a magenta sphere. D161 in Switch I is also indicated. (c) Interactions of the magnesium ion with the switch loops of NifH-ADP-AlF4. The colouring scheme is the same as in (B). D43 and D39 are indicated.
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fig5: The switch loops and Mg2+ co-ordination. (a) An overlap of the Clp1-ATP (green) and NifH-AlF4 (blue) structures around the P-loop, Switch I and switch II nucleotide binding regions. The backbone representation for each molecule is shown together with a stick representation for the ATP or ADP-AlF4. (b) Interactions of the magnesium ion with the switch loops of Clp1. Stick representations of the P-loop, Switch I and Switch II are shown in light red, yellow and blue, respectively. The adenine base of the ATP is shown in cyan and the magnesium ion is shown as a blue sphere. The water molecule co-ordinated by the magnesium ion and hydrogen bonded by the side-chain of Q164 together with α phosphate oxygen is shown as a magenta sphere. D161 in Switch I is also indicated. (c) Interactions of the magnesium ion with the switch loops of NifH-ADP-AlF4. The colouring scheme is the same as in (B). D43 and D39 are indicated.

Mentions: The P-loop, Switch I and Switch II loops of Clp1 display a degree of structural similarity with equivalent regions of NifH, illustrated by the structural overlap shown in Figure 5a. In NifH, residues in the switch loops do not contact the bound nucleotide directly but rather are involved in the co-ordination of the Mg2+ ion. Similarly, electron density that corresponds to an equivalent Mg2+ is present in the Clp1 structure, and residues in the switch loops are also involved in metal ion co-ordination. A result of this network of interactions is that the switch loops are brought together with the P-loop around the Mg2+ ion (Figure 5b). This network contains two axial ligands that coordinate the Mg2+ ion. These are a γ-phosphate oxygen together with the side-chain γ-oxygen of the P-loop T137. The side-chain of T137 also donates its hydrogen to the carboxylate of D251 in Switch II, effectively tethering together the Switch II and P-loops. This P-loop-Switch II interaction is a common feature conserved amongst many ATPases and GTPases but moreover the nature of the Mg2+ ion co-ordination in Clp1 is very similar to the Mg2+ co-ordination arrangement observed in the NifH–ADP–AlF4 − complex (Figure 5c). In Clp1, only two equatorial ligands, coordinating the Mg2+ ion are apparent, presumably due to the limited resolution. These equatorial ligands are β-phosphate oxygen and a single water molecule. In NifH–ADP–AlF4 − and in other SIMIBI-class ATPases where structures have been determined these same two interactions are conserved. However, in the NifH–ADP–AlF4 − complex two further equatorially positioned water molecules that make up the usual octahedral geometry of the magnesium co-ordination are also present. The three water molecules that are co-ordinated to the Mg2+ ion in NifH are hydrogen bonded by α-phosphate oxygen and by the main-chain and side-chains of residues in the Switch I and Switch II loops. In Clp1, the equatorially co-ordinated water molecule makes equivalent interactions as those observed for one of the NifH bound waters. In this case donating one hydrogen to the side-chain Oɛ1 of Q164 in the Switch I loop and the other to α-phosphate oxygen. Of the other potential Mg2+ ligands and/or hydrogen bond acceptors in the Switch regions the nearest are the carboxylate side-chain of the conserved D161 in Switch I and the main-chain carbonyl of T252 from the Switch II. In both cases the functional groups are located around 4 Å away from the Mg2+ ion so do not make interactions while the molecule is in this conformation. However, the equivalent residues in NifH, the D39 carboxylate and V126 carbonyl, mediate hydrogen bonding interactions with the two remaining equatorially liganded water molecules. Considering the structural similarity, it is likely that these residues have the same function in Clp1. Moreover, in the NifH–ADP–AlF4 − complex the carboxylate of D39 is hydrogen bonded to the in-line attacking water molecule indicating its importance in ATP hydrolysis and suggesting that D161 could perform the same catalytic function in Clp1.


Structure of a nucleotide-bound Clp1-Pcf11 polyadenylation factor.

Noble CG, Beuth B, Taylor IA - Nucleic Acids Res. (2006)

The switch loops and Mg2+ co-ordination. (a) An overlap of the Clp1-ATP (green) and NifH-AlF4 (blue) structures around the P-loop, Switch I and switch II nucleotide binding regions. The backbone representation for each molecule is shown together with a stick representation for the ATP or ADP-AlF4. (b) Interactions of the magnesium ion with the switch loops of Clp1. Stick representations of the P-loop, Switch I and Switch II are shown in light red, yellow and blue, respectively. The adenine base of the ATP is shown in cyan and the magnesium ion is shown as a blue sphere. The water molecule co-ordinated by the magnesium ion and hydrogen bonded by the side-chain of Q164 together with α phosphate oxygen is shown as a magenta sphere. D161 in Switch I is also indicated. (c) Interactions of the magnesium ion with the switch loops of NifH-ADP-AlF4. The colouring scheme is the same as in (B). D43 and D39 are indicated.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig5: The switch loops and Mg2+ co-ordination. (a) An overlap of the Clp1-ATP (green) and NifH-AlF4 (blue) structures around the P-loop, Switch I and switch II nucleotide binding regions. The backbone representation for each molecule is shown together with a stick representation for the ATP or ADP-AlF4. (b) Interactions of the magnesium ion with the switch loops of Clp1. Stick representations of the P-loop, Switch I and Switch II are shown in light red, yellow and blue, respectively. The adenine base of the ATP is shown in cyan and the magnesium ion is shown as a blue sphere. The water molecule co-ordinated by the magnesium ion and hydrogen bonded by the side-chain of Q164 together with α phosphate oxygen is shown as a magenta sphere. D161 in Switch I is also indicated. (c) Interactions of the magnesium ion with the switch loops of NifH-ADP-AlF4. The colouring scheme is the same as in (B). D43 and D39 are indicated.
Mentions: The P-loop, Switch I and Switch II loops of Clp1 display a degree of structural similarity with equivalent regions of NifH, illustrated by the structural overlap shown in Figure 5a. In NifH, residues in the switch loops do not contact the bound nucleotide directly but rather are involved in the co-ordination of the Mg2+ ion. Similarly, electron density that corresponds to an equivalent Mg2+ is present in the Clp1 structure, and residues in the switch loops are also involved in metal ion co-ordination. A result of this network of interactions is that the switch loops are brought together with the P-loop around the Mg2+ ion (Figure 5b). This network contains two axial ligands that coordinate the Mg2+ ion. These are a γ-phosphate oxygen together with the side-chain γ-oxygen of the P-loop T137. The side-chain of T137 also donates its hydrogen to the carboxylate of D251 in Switch II, effectively tethering together the Switch II and P-loops. This P-loop-Switch II interaction is a common feature conserved amongst many ATPases and GTPases but moreover the nature of the Mg2+ ion co-ordination in Clp1 is very similar to the Mg2+ co-ordination arrangement observed in the NifH–ADP–AlF4 − complex (Figure 5c). In Clp1, only two equatorial ligands, coordinating the Mg2+ ion are apparent, presumably due to the limited resolution. These equatorial ligands are β-phosphate oxygen and a single water molecule. In NifH–ADP–AlF4 − and in other SIMIBI-class ATPases where structures have been determined these same two interactions are conserved. However, in the NifH–ADP–AlF4 − complex two further equatorially positioned water molecules that make up the usual octahedral geometry of the magnesium co-ordination are also present. The three water molecules that are co-ordinated to the Mg2+ ion in NifH are hydrogen bonded by α-phosphate oxygen and by the main-chain and side-chains of residues in the Switch I and Switch II loops. In Clp1, the equatorially co-ordinated water molecule makes equivalent interactions as those observed for one of the NifH bound waters. In this case donating one hydrogen to the side-chain Oɛ1 of Q164 in the Switch I loop and the other to α-phosphate oxygen. Of the other potential Mg2+ ligands and/or hydrogen bond acceptors in the Switch regions the nearest are the carboxylate side-chain of the conserved D161 in Switch I and the main-chain carbonyl of T252 from the Switch II. In both cases the functional groups are located around 4 Å away from the Mg2+ ion so do not make interactions while the molecule is in this conformation. However, the equivalent residues in NifH, the D39 carboxylate and V126 carbonyl, mediate hydrogen bonding interactions with the two remaining equatorially liganded water molecules. Considering the structural similarity, it is likely that these residues have the same function in Clp1. Moreover, in the NifH–ADP–AlF4 − complex the carboxylate of D39 is hydrogen bonded to the in-line attacking water molecule indicating its importance in ATP hydrolysis and suggesting that D161 could perform the same catalytic function in Clp1.

Bottom Line: The arrangement of the nucleotide binding site is similar to that observed in SIMIBI-class ATPase subunits found in other multisubunit macromolecular complexes.However, despite this similarity, nucleotide hydrolysis does not occur.Moreover, we suggest that this complex represents a stabilized ATP bound form of Clp1 that requires the participation of other non-CFIA processing factors in order to initiate timely ATP hydrolysis during 3' end processing.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Structure, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.

ABSTRACT
Pcf11 and Clp1 are subunits of cleavage factor IA (CFIA), an essential polyadenylation factor in Saccahromyces cerevisiae. We have determined the structure of a ternary complex of Clp1 together with ATP and the Clp1-binding region of Pcf11. Clp1 contains three domains, a small N-terminal beta sandwich domain, a C-terminal domain containing a novel alpha/beta-fold and a central domain that binds ATP. The arrangement of the nucleotide binding site is similar to that observed in SIMIBI-class ATPase subunits found in other multisubunit macromolecular complexes. However, despite this similarity, nucleotide hydrolysis does not occur. The Pcf11 binding site is also located in the central domain where three highly conserved residues in Pcf11 mediate many of the protein-protein interactions. We propose that this conserved Clp1-Pcf11 interaction is responsible for maintaining a tight coupling between the Clp1 nucleotide binding subunit and the other components of the polyadenylation machinery. Moreover, we suggest that this complex represents a stabilized ATP bound form of Clp1 that requires the participation of other non-CFIA processing factors in order to initiate timely ATP hydrolysis during 3' end processing.

Show MeSH