Victor Bolaños - Academia.edu (original) (raw)
Papers by Victor Bolaños
Biochimica Et Biophysica Acta-general Subjects, 2006
Immobilised metal affinity chromatography (IMAC) is the most widely used technique for single-ste... more Immobilised metal affinity chromatography (IMAC) is the most widely used technique for single-step purification of recombinant proteins. However, despite its use in the purification of heterologue proteins in the eubacteria Escherichia coli for decades, the presence of native E. coli proteins that exhibit a high affinity for divalent cations such as nickel, cobalt or copper has remained problematic. This is of particular relevance when recombinant molecules are not expressed at high levels or when their overexpression induces that of native bacterial proteins due to pleiotropism and/or in response to stress conditions. Identification of such contaminating proteins is clearly relevant to those involved in the purification of histidine-tagged proteins either at small/medium scale or in high-throughput processes. The work presented here reviews the native proteins from E. coli most commonly co-purified by IMAC, including Fur, Crp, ArgE, SlyD, GlmS, GlgA, ODO1, ODO2, YadF and Yf bG. The binding of these proteins to metal-chelating resins can mostly be explained by their native metal-binding functions or their possession of surface clusters of histidine residues. However, some proteins fall outside these categories, implying that a further class of interactions may account for their ability to co-purify with histidine-tagged proteins. We propose a classification of these E. coli native proteins based on their physicochemical, structural and functional properties.
Structural Chemistry, 2002
Signaling in living systems needs to achieve high specificity, to be reversible, and to achieve h... more Signaling in living systems needs to achieve high specificity, to be reversible, and to achieve high signal to noise. Signaling mediated by multiprotein systems has evolved that avoids the requirement for high-affinity binary complexes that would be difficult to reverse and which, in the overcrowded cell, would lead to excessive noise in the system. Symmetrical structures are only occasionally formed. When they are, it is principally to colocate components, for example, the tyrosyl kinases of growth factors, where dimers form. Symmetry is, however, often broken, presumably to create more sensitivity and specificity in the signaling system by assembling other components, into higher-order multiprotein systems. The binding of a single heparin to two 1:1 FGF:FGFR complexes is an example, as is the binding of a single ligase to the Xrcc4 dimer, perhaps so creating a further DNA-binding site.
Acta Crystallographica Section D-biological Crystallography, 2004
A truncated form of the regulatory subunit of the protein kinase CK2 (residues 1±178) has been cr... more A truncated form of the regulatory subunit of the protein kinase CK2 (residues 1±178) has been crystallized in the presence of a fragment of the cyclin-dependent kinase inhibitor p21 WAF1 (residues 46±65) and the structure solved at 2.9 A Ê resolution by molecular replacement. The core of the CK2 dimer shows a high structural similarity with that identi®ed in previous structural analyses of the dimer and the holoenzyme. However, the electron density corresponding to the substrate-binding acidic loop (residues 55±64) indicates two conformations that differ from that of the holoenzyme structure , EMBO J. 20, 5320±5331]. Difference electron density near the dimerization region in each of the eight protomers in the asymmetric unit is attributed to between one and eight amino-acid residues of a complexed fragment of p21 WAF1 . This binding site corresponds to the solvent-accessible part of the conserved zinc-®nger motif.
Trends in Biochemical Sciences, 2006
Casein kinase 2 (CK2) is probably the most ubiquitous serine/threonine kinase found in eukaryotes... more Casein kinase 2 (CK2) is probably the most ubiquitous serine/threonine kinase found in eukaryotes: it phosphorylates >300 cellular proteins, ranging from transcription factors to proteins involved in chromatin structure and cell division. CK2 is a heterotetrameric enzyme that induces neoplastic growth when overexpressed. The b subunit of CK2 (CK2b) functions as the regulator of the catalytic CK2a and CK2a 0 subunits, enhancing their stability, activity and specificity. However, CK2b also functions as a multisubstrate docking platform for several other binding partners. Here, we discuss the organization and roles of interaction motifs of CK2b, postulate new protein-interaction sites and map these to the known interaction motifs, and show how the resulting complexity of interactions mediated by CK2 gives rise to the versatile functions of this pleiotropic protein kinase.
Recombinant yeast BUB1 N-terminal region residues 29-230 (Sc-BUB1 ) fused to an N-terminal GST ta... more Recombinant yeast BUB1 N-terminal region residues 29-230 (Sc-BUB1 ) fused to an N-terminal GST tag was expressed in E. coli BL21(DE3) cells according to reported protocols (Bolanos-Garcia et al., 2005). The expression protocol was modified for incorporation of L-selenomethionine (SeMet) into the protein as follows. A seed culture was prepared in LB medium (20 mL) and grew overnight at 37 o C. 1 mL of this culture was used to inoculate 250 mL of M9 broth prepared in 2 L baffled flasks that were previously equilibrated at the same temperature. M9 medium was supplemented with 1 mL of 4.2 g/L Fe 2 (SO) 4 (sterile filtered), 1 mL of MgSO 4 at 1M concentration (autoclaved), 10 mL of glucose at 40% (w/v) (sterile filtered) and 100 µL of 0.5% (w/v) thiamine (sterile filtered) per litre of culture. Growth was continued until absorbance at 600 nm was 0.3. At this point, a solution of L-Lysine, L-Phenylalanine and L-Threonine (all at 100 mg/L) as well as L-Isoleucine, L-Leucine, L-Valine and L-Selenomethionine (all at 50 mg/L) was added to the cultures. After 15 min for inhibition of Met synthesis to start, protein expression was induced with 1 mM IPTG. After 6 hr of growth, cell were harvested, flash-frozen in liquid nitrogen and stored at -80 o C.
Progress in Biophysics & Molecular Biology, 2009
This final part on 'perspectives' is focused on new strategies that can be used to crystallise pr... more This final part on 'perspectives' is focused on new strategies that can be used to crystallise proteins and improve the crystal quality of macromolecular complexes using any of the methods reviewed in this focused issue. Some advantages and disadvantages, limitations, and plausible applications to highresolution X-ray crystallography are discussed.
Structure, 2009
The interaction of the central mitotic checkpoint component BUB1 with the mitotic kinetochore pro... more The interaction of the central mitotic checkpoint component BUB1 with the mitotic kinetochore protein Blinkin is required for the kinetochore localization and function of BUB1 in the mitotic spindle assembly checkpoint, the regulatory mechanism of the cell cycle that ensures the even distribution of chromosomes during the transition from metaphase to anaphase. Here, we report the 1.74 Å resolution crystal structure of the N-terminal region of BUB1. The structure is organized as a tandem arrangement of three divergent units of the tetratricopeptide motif. Functional assays in vivo of native and site-specific mutants identify the residues of human BUB1 important for the interaction with Blinkin and define one region of potential therapeutic interest. The structure provides insight into the molecular basis of Blinkin-specific recognition by BUB1 and, on a broader perspective, of the mechanism that mediates kinetochore localization of BUB1 in checkpoint-activated cells.
Journal of Nucleic Acids, 2010
Nonhomologous end joining (NHEJ) plays a major role in double-strand break DNA repair, which invo... more Nonhomologous end joining (NHEJ) plays a major role in double-strand break DNA repair, which involves a series of steps mediated by multiprotein complexes. A ring-shaped Ku70/Ku80 heterodimer forms first at broken DNA ends, DNA-dependent protein kinase catalytic subunit (DNA-PKcs) binds to mediate synapsis and nucleases process DNA overhangs. DNA ligase IV (LigIV) is recruited as a complex with XRCC4 for ligation, with XLF/Cernunnos, playing a role in enhancing activity of LigIV. We describe how a combination of methods-X-ray crystallography, electron microscopy and small angle X-ray scatteringcan give insights into the transient multicomponent complexes that mediate NHEJ. We first consider the organisation of DNA-PKcs/Ku70/Ku80/DNA complex (DNA-PK) and then discuss emerging evidence concerning LigIV/XRCC4/XLF/DNA and higherorder complexes. We conclude by discussing roles of multiprotein systems in maintaining high signal-to-noise and the value of structural studies in developing new therapies in oncology and elsewhere.
Trends in Biochemical Sciences, 2011
The multidomain protein kinases BUB1 and BUBR1 (Mad3 in yeast, worms and plants) are central comp... more The multidomain protein kinases BUB1 and BUBR1 (Mad3 in yeast, worms and plants) are central components of the mitotic checkpoint for spindle assembly (SAC). This evolutionarily conserved and essential selfmonitoring system of the eukaryotic cell cycle ensures the high fidelity of chromosome segregation by delaying the onset of anaphase until all chromosomes are properly bi-oriented on the mitotic spindle. Despite their amino acid sequence conservation and similar domain organization, BUB1 and BUBR1 perform different functions in the SAC. Recent structural information provides crucial molecular insights into the regulation and recognition of BUB1 and BUBR1, and a solid foundation to dissect the roles of these proteins in the control of chromosome segregation in normal and oncogenic cells.
Embo Journal, 2008
The recently characterised 299-residue human XLF/ Cernunnos protein plays a crucial role in DNA r... more The recently characterised 299-residue human XLF/ Cernunnos protein plays a crucial role in DNA repair by non-homologous end joining (NHEJ) and interacts with the XRCC4-DNA Ligase IV complex. Here, we report the crystal structure of the XLF (1-233) homodimer at 2.3 Å resolution, confirming the predicted structural similarity to XRCC4. The XLF coiled-coil, however, is shorter than that of XRCC4 and undergoes an unexpected reverse in direction giving rise to a short distorted four helical bundle and a C-terminal helical structure wedged between the coiled-coil and head domain. The existence of a dimer as the major species is confirmed by size-exclusion chromatography, analytical ultracentrifugation, small-angle X-ray scattering and other biophysical methods. We show that the XLF structure is not easily compatible with a proposed XRCC4:XLF heterodimer. However, we demonstrate interactions between dimers of XLF and XRCC4 by surface plasmon resonance and analyse these in terms of surface properties, amino-acid conservation and mutations in immunodeficient patients. Our data are most consistent with head-to-head interactions in a 2:2:1 XRCC4:XLF:Ligase IV complex. EMBO THE EMBO JOURNAL THE Crystal structure of XLF/Cernunnos Y Li et al &
Journal of Crystal Growth, 2003
A method is described to control nucleation and coarsening in the process of protein crystallizat... more A method is described to control nucleation and coarsening in the process of protein crystallization. The control of these events is achieved by combining a biphasic oil/water system with a multi-compartment crystallization device. The addition of oil resulted in a significant decrease of the diffusion rate of a precipitant agent solution. When compared with crystal growth in the absence of oil, significantly smaller amounts of initially precipitated protein were observed. Thus, this method shifts crystal growth from a nucleation-coarsening (Ostwald ripening) equilibrium to a new equilibrium where Ostwald ripening is drastically diminished. Moreover, larger, rod-shape, high quality single crystals can also be obtained as a result of this equilibrium shift. r
Biochimica Et Biophysica Acta-general Subjects, 2006
Immobilised metal affinity chromatography (IMAC) is the most widely used technique for single-ste... more Immobilised metal affinity chromatography (IMAC) is the most widely used technique for single-step purification of recombinant proteins. However, despite its use in the purification of heterologue proteins in the eubacteria Escherichia coli for decades, the presence of native E. coli proteins that exhibit a high affinity for divalent cations such as nickel, cobalt or copper has remained problematic. This is of particular relevance when recombinant molecules are not expressed at high levels or when their overexpression induces that of native bacterial proteins due to pleiotropism and/or in response to stress conditions. Identification of such contaminating proteins is clearly relevant to those involved in the purification of histidine-tagged proteins either at small/medium scale or in high-throughput processes. The work presented here reviews the native proteins from E. coli most commonly co-purified by IMAC, including Fur, Crp, ArgE, SlyD, GlmS, GlgA, ODO1, ODO2, YadF and Yf bG. The binding of these proteins to metal-chelating resins can mostly be explained by their native metal-binding functions or their possession of surface clusters of histidine residues. However, some proteins fall outside these categories, implying that a further class of interactions may account for their ability to co-purify with histidine-tagged proteins. We propose a classification of these E. coli native proteins based on their physicochemical, structural and functional properties.
Structural Chemistry, 2002
Signaling in living systems needs to achieve high specificity, to be reversible, and to achieve h... more Signaling in living systems needs to achieve high specificity, to be reversible, and to achieve high signal to noise. Signaling mediated by multiprotein systems has evolved that avoids the requirement for high-affinity binary complexes that would be difficult to reverse and which, in the overcrowded cell, would lead to excessive noise in the system. Symmetrical structures are only occasionally formed. When they are, it is principally to colocate components, for example, the tyrosyl kinases of growth factors, where dimers form. Symmetry is, however, often broken, presumably to create more sensitivity and specificity in the signaling system by assembling other components, into higher-order multiprotein systems. The binding of a single heparin to two 1:1 FGF:FGFR complexes is an example, as is the binding of a single ligase to the Xrcc4 dimer, perhaps so creating a further DNA-binding site.
Acta Crystallographica Section D-biological Crystallography, 2004
A truncated form of the regulatory subunit of the protein kinase CK2 (residues 1±178) has been cr... more A truncated form of the regulatory subunit of the protein kinase CK2 (residues 1±178) has been crystallized in the presence of a fragment of the cyclin-dependent kinase inhibitor p21 WAF1 (residues 46±65) and the structure solved at 2.9 A Ê resolution by molecular replacement. The core of the CK2 dimer shows a high structural similarity with that identi®ed in previous structural analyses of the dimer and the holoenzyme. However, the electron density corresponding to the substrate-binding acidic loop (residues 55±64) indicates two conformations that differ from that of the holoenzyme structure , EMBO J. 20, 5320±5331]. Difference electron density near the dimerization region in each of the eight protomers in the asymmetric unit is attributed to between one and eight amino-acid residues of a complexed fragment of p21 WAF1 . This binding site corresponds to the solvent-accessible part of the conserved zinc-®nger motif.
Trends in Biochemical Sciences, 2006
Casein kinase 2 (CK2) is probably the most ubiquitous serine/threonine kinase found in eukaryotes... more Casein kinase 2 (CK2) is probably the most ubiquitous serine/threonine kinase found in eukaryotes: it phosphorylates >300 cellular proteins, ranging from transcription factors to proteins involved in chromatin structure and cell division. CK2 is a heterotetrameric enzyme that induces neoplastic growth when overexpressed. The b subunit of CK2 (CK2b) functions as the regulator of the catalytic CK2a and CK2a 0 subunits, enhancing their stability, activity and specificity. However, CK2b also functions as a multisubstrate docking platform for several other binding partners. Here, we discuss the organization and roles of interaction motifs of CK2b, postulate new protein-interaction sites and map these to the known interaction motifs, and show how the resulting complexity of interactions mediated by CK2 gives rise to the versatile functions of this pleiotropic protein kinase.
Recombinant yeast BUB1 N-terminal region residues 29-230 (Sc-BUB1 ) fused to an N-terminal GST ta... more Recombinant yeast BUB1 N-terminal region residues 29-230 (Sc-BUB1 ) fused to an N-terminal GST tag was expressed in E. coli BL21(DE3) cells according to reported protocols (Bolanos-Garcia et al., 2005). The expression protocol was modified for incorporation of L-selenomethionine (SeMet) into the protein as follows. A seed culture was prepared in LB medium (20 mL) and grew overnight at 37 o C. 1 mL of this culture was used to inoculate 250 mL of M9 broth prepared in 2 L baffled flasks that were previously equilibrated at the same temperature. M9 medium was supplemented with 1 mL of 4.2 g/L Fe 2 (SO) 4 (sterile filtered), 1 mL of MgSO 4 at 1M concentration (autoclaved), 10 mL of glucose at 40% (w/v) (sterile filtered) and 100 µL of 0.5% (w/v) thiamine (sterile filtered) per litre of culture. Growth was continued until absorbance at 600 nm was 0.3. At this point, a solution of L-Lysine, L-Phenylalanine and L-Threonine (all at 100 mg/L) as well as L-Isoleucine, L-Leucine, L-Valine and L-Selenomethionine (all at 50 mg/L) was added to the cultures. After 15 min for inhibition of Met synthesis to start, protein expression was induced with 1 mM IPTG. After 6 hr of growth, cell were harvested, flash-frozen in liquid nitrogen and stored at -80 o C.
Progress in Biophysics & Molecular Biology, 2009
This final part on 'perspectives' is focused on new strategies that can be used to crystallise pr... more This final part on 'perspectives' is focused on new strategies that can be used to crystallise proteins and improve the crystal quality of macromolecular complexes using any of the methods reviewed in this focused issue. Some advantages and disadvantages, limitations, and plausible applications to highresolution X-ray crystallography are discussed.
Structure, 2009
The interaction of the central mitotic checkpoint component BUB1 with the mitotic kinetochore pro... more The interaction of the central mitotic checkpoint component BUB1 with the mitotic kinetochore protein Blinkin is required for the kinetochore localization and function of BUB1 in the mitotic spindle assembly checkpoint, the regulatory mechanism of the cell cycle that ensures the even distribution of chromosomes during the transition from metaphase to anaphase. Here, we report the 1.74 Å resolution crystal structure of the N-terminal region of BUB1. The structure is organized as a tandem arrangement of three divergent units of the tetratricopeptide motif. Functional assays in vivo of native and site-specific mutants identify the residues of human BUB1 important for the interaction with Blinkin and define one region of potential therapeutic interest. The structure provides insight into the molecular basis of Blinkin-specific recognition by BUB1 and, on a broader perspective, of the mechanism that mediates kinetochore localization of BUB1 in checkpoint-activated cells.
Journal of Nucleic Acids, 2010
Nonhomologous end joining (NHEJ) plays a major role in double-strand break DNA repair, which invo... more Nonhomologous end joining (NHEJ) plays a major role in double-strand break DNA repair, which involves a series of steps mediated by multiprotein complexes. A ring-shaped Ku70/Ku80 heterodimer forms first at broken DNA ends, DNA-dependent protein kinase catalytic subunit (DNA-PKcs) binds to mediate synapsis and nucleases process DNA overhangs. DNA ligase IV (LigIV) is recruited as a complex with XRCC4 for ligation, with XLF/Cernunnos, playing a role in enhancing activity of LigIV. We describe how a combination of methods-X-ray crystallography, electron microscopy and small angle X-ray scatteringcan give insights into the transient multicomponent complexes that mediate NHEJ. We first consider the organisation of DNA-PKcs/Ku70/Ku80/DNA complex (DNA-PK) and then discuss emerging evidence concerning LigIV/XRCC4/XLF/DNA and higherorder complexes. We conclude by discussing roles of multiprotein systems in maintaining high signal-to-noise and the value of structural studies in developing new therapies in oncology and elsewhere.
Trends in Biochemical Sciences, 2011
The multidomain protein kinases BUB1 and BUBR1 (Mad3 in yeast, worms and plants) are central comp... more The multidomain protein kinases BUB1 and BUBR1 (Mad3 in yeast, worms and plants) are central components of the mitotic checkpoint for spindle assembly (SAC). This evolutionarily conserved and essential selfmonitoring system of the eukaryotic cell cycle ensures the high fidelity of chromosome segregation by delaying the onset of anaphase until all chromosomes are properly bi-oriented on the mitotic spindle. Despite their amino acid sequence conservation and similar domain organization, BUB1 and BUBR1 perform different functions in the SAC. Recent structural information provides crucial molecular insights into the regulation and recognition of BUB1 and BUBR1, and a solid foundation to dissect the roles of these proteins in the control of chromosome segregation in normal and oncogenic cells.
Embo Journal, 2008
The recently characterised 299-residue human XLF/ Cernunnos protein plays a crucial role in DNA r... more The recently characterised 299-residue human XLF/ Cernunnos protein plays a crucial role in DNA repair by non-homologous end joining (NHEJ) and interacts with the XRCC4-DNA Ligase IV complex. Here, we report the crystal structure of the XLF (1-233) homodimer at 2.3 Å resolution, confirming the predicted structural similarity to XRCC4. The XLF coiled-coil, however, is shorter than that of XRCC4 and undergoes an unexpected reverse in direction giving rise to a short distorted four helical bundle and a C-terminal helical structure wedged between the coiled-coil and head domain. The existence of a dimer as the major species is confirmed by size-exclusion chromatography, analytical ultracentrifugation, small-angle X-ray scattering and other biophysical methods. We show that the XLF structure is not easily compatible with a proposed XRCC4:XLF heterodimer. However, we demonstrate interactions between dimers of XLF and XRCC4 by surface plasmon resonance and analyse these in terms of surface properties, amino-acid conservation and mutations in immunodeficient patients. Our data are most consistent with head-to-head interactions in a 2:2:1 XRCC4:XLF:Ligase IV complex. EMBO THE EMBO JOURNAL THE Crystal structure of XLF/Cernunnos Y Li et al &
Journal of Crystal Growth, 2003
A method is described to control nucleation and coarsening in the process of protein crystallizat... more A method is described to control nucleation and coarsening in the process of protein crystallization. The control of these events is achieved by combining a biphasic oil/water system with a multi-compartment crystallization device. The addition of oil resulted in a significant decrease of the diffusion rate of a precipitant agent solution. When compared with crystal growth in the absence of oil, significantly smaller amounts of initially precipitated protein were observed. Thus, this method shifts crystal growth from a nucleation-coarsening (Ostwald ripening) equilibrium to a new equilibrium where Ostwald ripening is drastically diminished. Moreover, larger, rod-shape, high quality single crystals can also be obtained as a result of this equilibrium shift. r