Protein phosphatase 2A （PP2A） accounts for the majority of total Ser/Thr phosphatase activities in most cell types and regulates many biological processes. PP2A holoenzymes contain a scaffold A subunit, a cat- alyti...Protein phosphatase 2A （PP2A） accounts for the majority of total Ser/Thr phosphatase activities in most cell types and regulates many biological processes. PP2A holoenzymes contain a scaffold A subunit, a cat- alytic C subunit, and one of the regulatory/targeting B subunits. How the B subunit controls PP2A localization and substrate specificity, which is a crucial aspect of PP2A regulation, remains poorly understood. The kine- tochore is a critical site for PP2A functioning, where PP2A orchestrates chromosome segregation through its interactions with BubR1. The PP2A-BubR1 interac- tion plays important roles in both spindle checkpoint silencing and stable microtubule-kinetochore attach- ment. Here we present the crystal structure of a PP2A B56-BubRI complex, which demonstrates that a con- served BubRl LxxlxE motif binds to the concave side of the B56 pseudo-HEAT repeats. The BubR1 motif binds to a groove formed between B56 HEAT repeats 3 and 4, which is quite distant from the B56 binding surface for PP2A catalytic C subunit and thus is unlikely to affect PP2A activity. In addition, the BubR1 binding site on B56 is far from the B56 binding site of shugoshin, another kinetochore PP2A-binding protein, and thus BubR1 and shugoshin can potentially interact with PP2A-B56 simultaneously. Our structural and biochemical analysis indicates that other proteins with the LxxlxE motif may also bind to the same PP2A B56 surface. Thus, our structure of the PP2A B56-BubR1 complex provides important insights into how the B56 subunit directs the recruitment of PP2A to specific targets.展开更多
The important and diverse regulatory roles of Ca2+in eukaryotes are conveyed by the EF-hand containing calmodulin superfamily.However,the calcium-regulatory proteins in prokaryotes are still poorly understood.In this ...The important and diverse regulatory roles of Ca2+in eukaryotes are conveyed by the EF-hand containing calmodulin superfamily.However,the calcium-regulatory proteins in prokaryotes are still poorly understood.In this study,we report the three-dimensional structure of the calcium-binding protein from Streptomyces coelicolor,named CabD,which shares low sequence homology with other known helix-loop-helix EF-hand proteins.The CabD structure should provide insights into the biological role of the prokaryotic calcium-binding proteins.The unusual structural features of CabD compared with prokaryotic EF-hand proteins and eukaryotic sarcoplasmic calcium-binding proteins,including the bending conformation of the first C-terminalα-helix,unpaired ligand-binding EF-hands and the lack of the extreme Cterminal loop region,suggest it may have a distinct and significant function in calcium-mediated bacterial physiological processes,and provide a structural basis for potential calcium-mediated regulatory roles in prokaryotes.展开更多
Sometimes crystals cannot diffract X-rays beyond 3.0Åresolution due to the intrinsic flexibility associated with the protein.Low resolution diffraction data not only pose a challenge to structure determination,bu...Sometimes crystals cannot diffract X-rays beyond 3.0Åresolution due to the intrinsic flexibility associated with the protein.Low resolution diffraction data not only pose a challenge to structure determination,but also hamper interpretation of mechanistic details.Crystals of a 25.6 kDa non-Pfam,hypothetical protein,PF2046,diffracted X-rays to 3.38Åresolution.A combination of SeMet derived heavy atom positions with multiple cycles of B-factor sharpening,multi-crystal averaging,restrained refinement followed by manual inspection of electron density and model building resulted in a final model with a R value of 23.5(R_(free)=24.7).The asymmetric unit was large and consisted of six molecules arranged as a homodimer of trimers.Analysis of the structure revealed the presence of a RNA binding domain suggesting a role for PF2046 in the processing of nucleic acids.展开更多
文摘Protein phosphatase 2A （PP2A） accounts for the majority of total Ser/Thr phosphatase activities in most cell types and regulates many biological processes. PP2A holoenzymes contain a scaffold A subunit, a cat- alytic C subunit, and one of the regulatory/targeting B subunits. How the B subunit controls PP2A localization and substrate specificity, which is a crucial aspect of PP2A regulation, remains poorly understood. The kine- tochore is a critical site for PP2A functioning, where PP2A orchestrates chromosome segregation through its interactions with BubR1. The PP2A-BubR1 interac- tion plays important roles in both spindle checkpoint silencing and stable microtubule-kinetochore attach- ment. Here we present the crystal structure of a PP2A B56-BubRI complex, which demonstrates that a con- served BubRl LxxlxE motif binds to the concave side of the B56 pseudo-HEAT repeats. The BubR1 motif binds to a groove formed between B56 HEAT repeats 3 and 4, which is quite distant from the B56 binding surface for PP2A catalytic C subunit and thus is unlikely to affect PP2A activity. In addition, the BubR1 binding site on B56 is far from the B56 binding site of shugoshin, another kinetochore PP2A-binding protein, and thus BubR1 and shugoshin can potentially interact with PP2A-B56 simultaneously. Our structural and biochemical analysis indicates that other proteins with the LxxlxE motif may also bind to the same PP2A B56 surface. Thus, our structure of the PP2A B56-BubR1 complex provides important insights into how the B56 subunit directs the recruitment of PP2A to specific targets.
基金supported by the National Natural Science Foundation of China(Grant Nos.30400259,30221003)the National Basic Research Program(973 Program)(Grant No.2007CB914301)the Tianjin Municipal Science and Technology Commission(Grant No.08SYSYTC00200).
文摘The important and diverse regulatory roles of Ca2+in eukaryotes are conveyed by the EF-hand containing calmodulin superfamily.However,the calcium-regulatory proteins in prokaryotes are still poorly understood.In this study,we report the three-dimensional structure of the calcium-binding protein from Streptomyces coelicolor,named CabD,which shares low sequence homology with other known helix-loop-helix EF-hand proteins.The CabD structure should provide insights into the biological role of the prokaryotic calcium-binding proteins.The unusual structural features of CabD compared with prokaryotic EF-hand proteins and eukaryotic sarcoplasmic calcium-binding proteins,including the bending conformation of the first C-terminalα-helix,unpaired ligand-binding EF-hands and the lack of the extreme Cterminal loop region,suggest it may have a distinct and significant function in calcium-mediated bacterial physiological processes,and provide a structural basis for potential calcium-mediated regulatory roles in prokaryotes.
基金This work was funded by the Ministry of Science and Technology of China(Grant Nos.2006AA02A316,2009DFB30310 and 2006CB910901)the National Natural Science Foundation of China(Grants Nos.30670427 and 30721003)+1 种基金the Ministry of Health of China(Grant No.2008ZX10404)CAS Research Grant(No.KSCX2-YW-R-127 and INFO-115-D01-2009).
文摘Sometimes crystals cannot diffract X-rays beyond 3.0Åresolution due to the intrinsic flexibility associated with the protein.Low resolution diffraction data not only pose a challenge to structure determination,but also hamper interpretation of mechanistic details.Crystals of a 25.6 kDa non-Pfam,hypothetical protein,PF2046,diffracted X-rays to 3.38Åresolution.A combination of SeMet derived heavy atom positions with multiple cycles of B-factor sharpening,multi-crystal averaging,restrained refinement followed by manual inspection of electron density and model building resulted in a final model with a R value of 23.5(R_(free)=24.7).The asymmetric unit was large and consisted of six molecules arranged as a homodimer of trimers.Analysis of the structure revealed the presence of a RNA binding domain suggesting a role for PF2046 in the processing of nucleic acids.
