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The crystal structure of the RhoA-AKAP-Lbc DH-PH domain complex.

Abdul Azeez KR, Knapp S, Fernandes JM, Klussmann E, Elkins JM - Biochem. J. (2014)

Bottom Line: The structure reveals important differences compared with related RhoGEF proteins such as leukaemia-associated RhoGEF.Comparison with a structure of the isolated AKAP-Lbc DH domain revealed a change in conformation of the N-terminal 'GEF switch' region upon binding to RhoA.Isothermal titration calorimetry showed that AKAP-Lbc has only micromolar affinity for RhoA, which combined with the presence of potential binding pockets for small molecules on AKAP-Lbc, raises the possibility of targeting AKAP-Lbc with GEF inhibitors.

View Article: PubMed Central - PubMed

Affiliation: *Structural Genomics Consortium, Oxford University, Old Road Campus Research Building, Old Road Campus, Roosevelt Drive, Oxford OX3 7DQ, U.K.

ABSTRACT
The RhoGEF (Rho GTPase guanine-nucleotide-exchange factor) domain of AKAP-Lbc (A-kinase-anchoring protein-Lbc, also known as AKAP13) catalyses nucleotide exchange on RhoA and is involved in the development of cardiac hypertrophy. The RhoGEF activity of AKAP-Lbc has also been implicated in cancer. We have determined the X-ray crystal structure of the complex between RhoA-GDP and the AKAP-Lbc RhoGEF [DH (Dbl-homologous)-PH (pleckstrin homology)] domain to 2.1 Å (1 Å = 0.1 nm) resolution. The structure reveals important differences compared with related RhoGEF proteins such as leukaemia-associated RhoGEF. Nucleotide-exchange assays comparing the activity of the DH-PH domain to the DH domain alone showed no role for the PH domain in nucleotide exchange, which is explained by the RhoA-AKAP-Lbc structure. Comparison with a structure of the isolated AKAP-Lbc DH domain revealed a change in conformation of the N-terminal 'GEF switch' region upon binding to RhoA. Isothermal titration calorimetry showed that AKAP-Lbc has only micromolar affinity for RhoA, which combined with the presence of potential binding pockets for small molecules on AKAP-Lbc, raises the possibility of targeting AKAP-Lbc with GEF inhibitors.

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Comparison of the GEF switch region at the N-terminus of the AKAP-Lbc DH domain with the equivalent regions in LARG and p115-RhoGEF(A) The conformation of the AKAP-Lbc DH domain GEF switch region (coloured in magenta) when bound to RhoA. RhoA is coloured with red α-helices and yellow β-strands with its switch I region in green. The DH domain of AKAP-Lbc is coloured blue. (B) The conformation of the DH domain when not bound to RhoA. (C and D) The conformation of the equivalent regions in LARG when bound to RhoA (C) and p115-RhoGEF when not bound to RhoA (D). (E) Sequence alignment of the GEF switch regions of AKAP-Lbc and three homologous RhoGEF domains. Residues important for the packing of the GEF switch region are marked with asterisks below the alignment.
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Figure 5: Comparison of the GEF switch region at the N-terminus of the AKAP-Lbc DH domain with the equivalent regions in LARG and p115-RhoGEF(A) The conformation of the AKAP-Lbc DH domain GEF switch region (coloured in magenta) when bound to RhoA. RhoA is coloured with red α-helices and yellow β-strands with its switch I region in green. The DH domain of AKAP-Lbc is coloured blue. (B) The conformation of the DH domain when not bound to RhoA. (C and D) The conformation of the equivalent regions in LARG when bound to RhoA (C) and p115-RhoGEF when not bound to RhoA (D). (E) Sequence alignment of the GEF switch regions of AKAP-Lbc and three homologous RhoGEF domains. Residues important for the packing of the GEF switch region are marked with asterisks below the alignment.

Mentions: The GEF switch region of AKAP-Lbc's DH domain (Figure 5A) adopts a similar conformation to that of LARG (Figure 5C). RhoA Tyr34 packs against the conserved Glu2001 (LARG Glu790), whereas forming a hydrogen bond to the also conserved Glu2005 (LARG Glu794). The tryptophan appears to orient the backbone of the GEF switch region so that Glu2001 can form hydrogen bonds to two backbone nitrogens (Figures 5A and 5C). This presumably stabilizes the conformation of Glu2001 in a conformation favourable for interaction with RhoA Tyr34.


The crystal structure of the RhoA-AKAP-Lbc DH-PH domain complex.

Abdul Azeez KR, Knapp S, Fernandes JM, Klussmann E, Elkins JM - Biochem. J. (2014)

Comparison of the GEF switch region at the N-terminus of the AKAP-Lbc DH domain with the equivalent regions in LARG and p115-RhoGEF(A) The conformation of the AKAP-Lbc DH domain GEF switch region (coloured in magenta) when bound to RhoA. RhoA is coloured with red α-helices and yellow β-strands with its switch I region in green. The DH domain of AKAP-Lbc is coloured blue. (B) The conformation of the DH domain when not bound to RhoA. (C and D) The conformation of the equivalent regions in LARG when bound to RhoA (C) and p115-RhoGEF when not bound to RhoA (D). (E) Sequence alignment of the GEF switch regions of AKAP-Lbc and three homologous RhoGEF domains. Residues important for the packing of the GEF switch region are marked with asterisks below the alignment.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4232260&req=5

Figure 5: Comparison of the GEF switch region at the N-terminus of the AKAP-Lbc DH domain with the equivalent regions in LARG and p115-RhoGEF(A) The conformation of the AKAP-Lbc DH domain GEF switch region (coloured in magenta) when bound to RhoA. RhoA is coloured with red α-helices and yellow β-strands with its switch I region in green. The DH domain of AKAP-Lbc is coloured blue. (B) The conformation of the DH domain when not bound to RhoA. (C and D) The conformation of the equivalent regions in LARG when bound to RhoA (C) and p115-RhoGEF when not bound to RhoA (D). (E) Sequence alignment of the GEF switch regions of AKAP-Lbc and three homologous RhoGEF domains. Residues important for the packing of the GEF switch region are marked with asterisks below the alignment.
Mentions: The GEF switch region of AKAP-Lbc's DH domain (Figure 5A) adopts a similar conformation to that of LARG (Figure 5C). RhoA Tyr34 packs against the conserved Glu2001 (LARG Glu790), whereas forming a hydrogen bond to the also conserved Glu2005 (LARG Glu794). The tryptophan appears to orient the backbone of the GEF switch region so that Glu2001 can form hydrogen bonds to two backbone nitrogens (Figures 5A and 5C). This presumably stabilizes the conformation of Glu2001 in a conformation favourable for interaction with RhoA Tyr34.

Bottom Line: The structure reveals important differences compared with related RhoGEF proteins such as leukaemia-associated RhoGEF.Comparison with a structure of the isolated AKAP-Lbc DH domain revealed a change in conformation of the N-terminal 'GEF switch' region upon binding to RhoA.Isothermal titration calorimetry showed that AKAP-Lbc has only micromolar affinity for RhoA, which combined with the presence of potential binding pockets for small molecules on AKAP-Lbc, raises the possibility of targeting AKAP-Lbc with GEF inhibitors.

View Article: PubMed Central - PubMed

Affiliation: *Structural Genomics Consortium, Oxford University, Old Road Campus Research Building, Old Road Campus, Roosevelt Drive, Oxford OX3 7DQ, U.K.

ABSTRACT
The RhoGEF (Rho GTPase guanine-nucleotide-exchange factor) domain of AKAP-Lbc (A-kinase-anchoring protein-Lbc, also known as AKAP13) catalyses nucleotide exchange on RhoA and is involved in the development of cardiac hypertrophy. The RhoGEF activity of AKAP-Lbc has also been implicated in cancer. We have determined the X-ray crystal structure of the complex between RhoA-GDP and the AKAP-Lbc RhoGEF [DH (Dbl-homologous)-PH (pleckstrin homology)] domain to 2.1 Å (1 Å = 0.1 nm) resolution. The structure reveals important differences compared with related RhoGEF proteins such as leukaemia-associated RhoGEF. Nucleotide-exchange assays comparing the activity of the DH-PH domain to the DH domain alone showed no role for the PH domain in nucleotide exchange, which is explained by the RhoA-AKAP-Lbc structure. Comparison with a structure of the isolated AKAP-Lbc DH domain revealed a change in conformation of the N-terminal 'GEF switch' region upon binding to RhoA. Isothermal titration calorimetry showed that AKAP-Lbc has only micromolar affinity for RhoA, which combined with the presence of potential binding pockets for small molecules on AKAP-Lbc, raises the possibility of targeting AKAP-Lbc with GEF inhibitors.

Show MeSH
Related in: MedlinePlus