José A Gavira | CSIC (Consejo Superior de Investigaciones Científicas-Spanish National Research Council) (original) (raw)
Papers by José A Gavira
Crystal Growth & Design
mBio
Although antagonists were found to bind different bacterial signal transduction receptors, we are... more Although antagonists were found to bind different bacterial signal transduction receptors, we are still at the early stages of understanding the molecular details by which these molecules exert their inhibitory effects. Here, we provide insight into the structural changes resulting from the binding of an agonist and an antagonist to a sensor protein.
Israel Journal of Chemistry
Enzymatic, mass spectrometry, crystallography and SAXS data of AKR1B1
7th International School on Biological Crystallization (ISBC2019), Granada (Spain), 26th-31st may
Crystal Growth & Design, 2019
Human Molecular Genetics, 2017
Human proteins are vulnerable towards disease-associated single amino acid replacements affecting... more Human proteins are vulnerable towards disease-associated single amino acid replacements affecting protein stability and function. Interestingly, a few studies have shown that consensus amino acids from mammals or vertebrates can enhance protein stability when incorporated into human proteins. Here, we investigate yet unexplored relationships between the high vulnerability of human proteins towards disease-associated inactivation and recent evolutionary site-specific divergence of stabilizing amino acids. Using phylogenetic, structural and experimental analyses, we show that divergence from the consensus amino acids at several sites during mammalian evolution has caused local protein destabilization in two human proteins linked to disease: cancer-associated NQO1 and alanine:glyoxylate aminotransferase, mutated in primary hyperoxaluria type I. We demonstrate that a single consensus mutation (H80R) acts as a disease suppressor on the most common cancerassociated polymorphism in NQO1 (P187S). The H80R mutation reactivates P187S by enhancing FAD binding affinity through local and dynamic stabilization of its binding site. Furthermore, we show how a second suppressor mutation (E247Q) cooperates with H80R in protecting the P187S polymorphism towards inactivation through long-range allosteric communication within the structural ensemble of the protein. Our results support that recent divergence of consensus amino acids may have occurred with neutral effects on many functional and regulatory traits of wild-type human proteins. However, divergence at †
Acta crystallographica, 2008
A recombinant active-site mutant of hydantoin racemase (C76A) from Sinorhizobium meliloti CECT 41... more A recombinant active-site mutant of hydantoin racemase (C76A) from Sinorhizobium meliloti CECT 4114 (SmeHyuA) has been crystallized in the presence and absence of the substrate d,l-5-isopropyl hydantoin. Crystals of the SmeHyuA mutant suitable for data collection and structure determination were grown using the counter-diffusion method. X-ray data were collected to resolutions of 2.17 and 1.85 Å for the free and bound enzymes, respectively. Both crystals belong to space group R3 and contain two molecules of SmeHyuA per asymmetric unit. The crystals of the free and complexed SmeHyuA have unit-cell parameters a = b = 85.43, c = 152.37 Å and a = b = 85.69, c = 154.38 Å , crystal volumes per protein weight (V M) of 1.94 and 1.98 Å 3 Da À1 and solvent contents of 36.7 and 37.9%, respectively.
Journal of Crystal Growth
ABSTRACT
The potential of crosslinked hydrogels in the crystallization of proteins is an area that has not... more The potential of crosslinked hydrogels in the crystallization of proteins is an area that has not been exploited. The technique of counter-diffusion is a novel method for the crystallization of proteins which entails the minimization of convection effects resulting in high quality crystals. The implementation of this technique in capillary tubes with different diameters has been accepted for the crystallization of proteins, by using a precipitating solution which diffuses through an agarose polymer that controls the diffusion process. The diffusional process using such systems can be controlled but cannot be widely manipulated. The use of crosslinked hydrogels provides better diffusional and environmental control. This research focuses on the role of crosslinked hydrogels on the nucleation process involved in protein crystallization. The crystallization process was performed using crosslinked Poly(ethyleneglycol) hydrogels and lysozyme as a model protein. The effects of polymer conc...
Proceedings of the National Academy of Sciences, 2012
Chemoreceptor-based signaling is a central mechanism in bacterial signal transduction. Receptors ... more Chemoreceptor-based signaling is a central mechanism in bacterial signal transduction. Receptors are classified according to the size of their ligand-binding region. The well-studied cluster I proteins have a 100- to 150-residue ligand-binding region that contains a single site for chemoattractant recognition. Cluster II receptors, which contain a 220- to 300-residue ligand-binding region and which are almost as abundant as cluster I receptors, remain largely uncharacterized. Here, we report high-resolution structures of the ligand-binding region of the cluster II McpS chemotaxis receptor (McpS-LBR) of Pseudomonas putida KT2440 in complex with different chemoattractants. The structure of McpS-LBR represents a small-molecule binding domain composed of two modules, each able to bind different signal molecules. Malate and succinate were found to bind to the membrane-proximal module, whereas acetate binds to the membrane-distal module. A structural alignment of the two modules revealed ...
Journal of Crystal Growth, 2003
We have analyzed the crystal quality along a capillary by a precise protocol that comprises the s... more We have analyzed the crystal quality along a capillary by a precise protocol that comprises the study of tetragonal lysozyme cylindrical crystals that fill the capillary diameter (i.e. rods), the careful definition of the diffraction parameters and the use of a single software for the data reduction in order to avoid any bias in the comparison of the quality of different data sets. Our results cannot be explained on the basis of the different redundancy of the data sets and they demonstrate that the gel acupuncture method promotes a gradient of supersaturation along the capillary that yields in the same experiment crystals of increasing quality as a function of the position. However, despite being single crystals, rods have regions that show different crystal quality because they grew at different supersaturations. Our data are in agreement with the existence of a relation between length of the c-axis and crystal quality reported by other groups, but a deeper analysis of the cell parameters reveals the existence of a significant linear relation (R ¼ 0:87) with the c/a-axis ratio. This result points to the hypothesis of an ideal unit cell that yields the best crystals in terms of I=sðIÞ:
Journal of Bacteriology, 2012
N -Carbamoyl- l -amino acid amidohydrolases ( l -carbamoylases) are important industrial enzymes ... more N -Carbamoyl- l -amino acid amidohydrolases ( l -carbamoylases) are important industrial enzymes used in kinetic resolution of racemic mixtures of N -carbamoyl-amino acids due to their strict enantiospecificity. In this work, we report the first l -carbamoylase structure belonging to Geobacillus stearothermophilus CECT43 (BsLcar), at a resolution of 2.7 Å. Structural analysis of BsLcar and several members of the peptidase M20/M25/M40 family confirmed the expected conserved residues at the active site in this family, and site-directed mutagenesis revealed their relevance to substrate binding. We also found an unexpectedly conserved arginine residue (Arg 234 in BsLcar), proven to be critical for dimerization of the enzyme. The mutation of this sole residue resulted in a total loss of activity and prevented the formation of the dimer in BsLcar. Comparative studies revealed that the dimerization domain of the peptidase M20/M25/M40 family is a “small-molecule binding domain,” allowing fu...
Crystal Growth & Design, 2011
ABSTRACT In protein crystallization trials there is always some ambiguity in defining if the nucl... more ABSTRACT In protein crystallization trials there is always some ambiguity in defining if the nucleation event occurred by a homogeneous or a heterogeneous process. One of the reasons comes from the difficulty to compare experiments with or without a heterogeneous nucleant under the same chemical conditions. The outcome is that it is difficult to figure out the values of supersaturation addressing homo- versus heterogeneous nucleation. Recently it has been shown that the crystallization mushroom allows performance of sitting drop vapor diffusion experiments under identical physical-chemical conditions and that functionalized surfaces are favorable heterogeneous substrates for protein crystallization. With the aim to discriminate between nucleation processes, three model proteins, lysozyme, glucose isomerase, and thaumatin, have been crystallized at different starting concentrations, using positively and negatively charged surfaces, in the crystallization mushroom. The results show that (i) the heterogenenous nucleation does not crucially affect the nucleation process under conditions of high supersaturation and that (ii) the nucleating action of the fianctionalized surfaces is mainly related to their superficial charge distribution more than to their absolute charge, according to both classical and colloidal nucleation theories.
Acta Crystallographica Section F Structural Biology and Crystallization Communications, 2012
Pseudomonas putidaPtxS is a member of the LacI protein family of transcriptional regulators invol... more Pseudomonas putidaPtxS is a member of the LacI protein family of transcriptional regulators involved in glucose metabolism. All genes involved in this pathway are clustered into two operons,kguandgad. PtxS controls the expression of thekguandgadoperons as well as its own transcription. The PtxS operator is a perfect palindrome, 5′-TGAAACCGGTTTCA-3′, which is present in all three promoters. Crystallization of native PtxS failed, and PtxS–DNA crystals were finally produced by the counter-diffusion technique. A portion of the capillary used for crystal growth was attached to the end of a SPINE standard cap and directly flash-cooled in liquid nitrogen for diffraction tests. A full data set was collected with a beam size of 10 × 10 µm. The crystal belonged to the trigonal space groupP3, with unit-cell parametersa=b= 213.71,c = 71.57 Å. Only unhandled crystals grown in capillaries of 0.1 mm inner diameter diffracted X-rays to 1.92 Å resolution.
Acta Crystallographica Section D Biological Crystallography, 2011
SH3 domains are small protein modules that mediate the assembly of specific protein complexes, ty... more SH3 domains are small protein modules that mediate the assembly of specific protein complexes, typically via binding to proline-rich sequences in their respective binding partners. Most of the α-spectrin SH3-domain (Spc-SH3) structures determined to date using X-ray diffraction have been solved from crystals belonging to the orthorhombic space group P2(1)2(1)2(1) with a needle-like morphology. All of these orthorhombic crystals exhibited a rapid growth rate. In addition to this crystal form, the R21D mutant of Spc-SH3 crystallizes in a new crystal form in the presence of sodium formate at pH values higher than 6. This new crystal form grows at a slower rate and belongs to the hexagonal space group P6(5)22, with unit-cell parameters a = b = 42.9, c = 127.5 Å. When both polymorphs of the R21D mutant of Spc-SH3 are simultaneously present into the same solution, it has been observed that the hexagonal crystals grow at the expense of the orthorhombic crystals. The availability of 1.1 Å resolution structures for both crystal forms allows the identification of key features that could account for the observed polymorphic behaviour.
Acta Crystallographica Section A Foundations of Crystallography, 2004
Acta Crystallographica Section A Foundations of Crystallography, 2004
Journal of Crystal Growth, 2001
ABSTRACT
International Journal of Biological Macromolecules
Crystal Growth & Design
mBio
Although antagonists were found to bind different bacterial signal transduction receptors, we are... more Although antagonists were found to bind different bacterial signal transduction receptors, we are still at the early stages of understanding the molecular details by which these molecules exert their inhibitory effects. Here, we provide insight into the structural changes resulting from the binding of an agonist and an antagonist to a sensor protein.
Israel Journal of Chemistry
Enzymatic, mass spectrometry, crystallography and SAXS data of AKR1B1
7th International School on Biological Crystallization (ISBC2019), Granada (Spain), 26th-31st may
Crystal Growth & Design, 2019
Human Molecular Genetics, 2017
Human proteins are vulnerable towards disease-associated single amino acid replacements affecting... more Human proteins are vulnerable towards disease-associated single amino acid replacements affecting protein stability and function. Interestingly, a few studies have shown that consensus amino acids from mammals or vertebrates can enhance protein stability when incorporated into human proteins. Here, we investigate yet unexplored relationships between the high vulnerability of human proteins towards disease-associated inactivation and recent evolutionary site-specific divergence of stabilizing amino acids. Using phylogenetic, structural and experimental analyses, we show that divergence from the consensus amino acids at several sites during mammalian evolution has caused local protein destabilization in two human proteins linked to disease: cancer-associated NQO1 and alanine:glyoxylate aminotransferase, mutated in primary hyperoxaluria type I. We demonstrate that a single consensus mutation (H80R) acts as a disease suppressor on the most common cancerassociated polymorphism in NQO1 (P187S). The H80R mutation reactivates P187S by enhancing FAD binding affinity through local and dynamic stabilization of its binding site. Furthermore, we show how a second suppressor mutation (E247Q) cooperates with H80R in protecting the P187S polymorphism towards inactivation through long-range allosteric communication within the structural ensemble of the protein. Our results support that recent divergence of consensus amino acids may have occurred with neutral effects on many functional and regulatory traits of wild-type human proteins. However, divergence at †
Acta crystallographica, 2008
A recombinant active-site mutant of hydantoin racemase (C76A) from Sinorhizobium meliloti CECT 41... more A recombinant active-site mutant of hydantoin racemase (C76A) from Sinorhizobium meliloti CECT 4114 (SmeHyuA) has been crystallized in the presence and absence of the substrate d,l-5-isopropyl hydantoin. Crystals of the SmeHyuA mutant suitable for data collection and structure determination were grown using the counter-diffusion method. X-ray data were collected to resolutions of 2.17 and 1.85 Å for the free and bound enzymes, respectively. Both crystals belong to space group R3 and contain two molecules of SmeHyuA per asymmetric unit. The crystals of the free and complexed SmeHyuA have unit-cell parameters a = b = 85.43, c = 152.37 Å and a = b = 85.69, c = 154.38 Å , crystal volumes per protein weight (V M) of 1.94 and 1.98 Å 3 Da À1 and solvent contents of 36.7 and 37.9%, respectively.
Journal of Crystal Growth
ABSTRACT
The potential of crosslinked hydrogels in the crystallization of proteins is an area that has not... more The potential of crosslinked hydrogels in the crystallization of proteins is an area that has not been exploited. The technique of counter-diffusion is a novel method for the crystallization of proteins which entails the minimization of convection effects resulting in high quality crystals. The implementation of this technique in capillary tubes with different diameters has been accepted for the crystallization of proteins, by using a precipitating solution which diffuses through an agarose polymer that controls the diffusion process. The diffusional process using such systems can be controlled but cannot be widely manipulated. The use of crosslinked hydrogels provides better diffusional and environmental control. This research focuses on the role of crosslinked hydrogels on the nucleation process involved in protein crystallization. The crystallization process was performed using crosslinked Poly(ethyleneglycol) hydrogels and lysozyme as a model protein. The effects of polymer conc...
Proceedings of the National Academy of Sciences, 2012
Chemoreceptor-based signaling is a central mechanism in bacterial signal transduction. Receptors ... more Chemoreceptor-based signaling is a central mechanism in bacterial signal transduction. Receptors are classified according to the size of their ligand-binding region. The well-studied cluster I proteins have a 100- to 150-residue ligand-binding region that contains a single site for chemoattractant recognition. Cluster II receptors, which contain a 220- to 300-residue ligand-binding region and which are almost as abundant as cluster I receptors, remain largely uncharacterized. Here, we report high-resolution structures of the ligand-binding region of the cluster II McpS chemotaxis receptor (McpS-LBR) of Pseudomonas putida KT2440 in complex with different chemoattractants. The structure of McpS-LBR represents a small-molecule binding domain composed of two modules, each able to bind different signal molecules. Malate and succinate were found to bind to the membrane-proximal module, whereas acetate binds to the membrane-distal module. A structural alignment of the two modules revealed ...
Journal of Crystal Growth, 2003
We have analyzed the crystal quality along a capillary by a precise protocol that comprises the s... more We have analyzed the crystal quality along a capillary by a precise protocol that comprises the study of tetragonal lysozyme cylindrical crystals that fill the capillary diameter (i.e. rods), the careful definition of the diffraction parameters and the use of a single software for the data reduction in order to avoid any bias in the comparison of the quality of different data sets. Our results cannot be explained on the basis of the different redundancy of the data sets and they demonstrate that the gel acupuncture method promotes a gradient of supersaturation along the capillary that yields in the same experiment crystals of increasing quality as a function of the position. However, despite being single crystals, rods have regions that show different crystal quality because they grew at different supersaturations. Our data are in agreement with the existence of a relation between length of the c-axis and crystal quality reported by other groups, but a deeper analysis of the cell parameters reveals the existence of a significant linear relation (R ¼ 0:87) with the c/a-axis ratio. This result points to the hypothesis of an ideal unit cell that yields the best crystals in terms of I=sðIÞ:
Journal of Bacteriology, 2012
N -Carbamoyl- l -amino acid amidohydrolases ( l -carbamoylases) are important industrial enzymes ... more N -Carbamoyl- l -amino acid amidohydrolases ( l -carbamoylases) are important industrial enzymes used in kinetic resolution of racemic mixtures of N -carbamoyl-amino acids due to their strict enantiospecificity. In this work, we report the first l -carbamoylase structure belonging to Geobacillus stearothermophilus CECT43 (BsLcar), at a resolution of 2.7 Å. Structural analysis of BsLcar and several members of the peptidase M20/M25/M40 family confirmed the expected conserved residues at the active site in this family, and site-directed mutagenesis revealed their relevance to substrate binding. We also found an unexpectedly conserved arginine residue (Arg 234 in BsLcar), proven to be critical for dimerization of the enzyme. The mutation of this sole residue resulted in a total loss of activity and prevented the formation of the dimer in BsLcar. Comparative studies revealed that the dimerization domain of the peptidase M20/M25/M40 family is a “small-molecule binding domain,” allowing fu...
Crystal Growth & Design, 2011
ABSTRACT In protein crystallization trials there is always some ambiguity in defining if the nucl... more ABSTRACT In protein crystallization trials there is always some ambiguity in defining if the nucleation event occurred by a homogeneous or a heterogeneous process. One of the reasons comes from the difficulty to compare experiments with or without a heterogeneous nucleant under the same chemical conditions. The outcome is that it is difficult to figure out the values of supersaturation addressing homo- versus heterogeneous nucleation. Recently it has been shown that the crystallization mushroom allows performance of sitting drop vapor diffusion experiments under identical physical-chemical conditions and that functionalized surfaces are favorable heterogeneous substrates for protein crystallization. With the aim to discriminate between nucleation processes, three model proteins, lysozyme, glucose isomerase, and thaumatin, have been crystallized at different starting concentrations, using positively and negatively charged surfaces, in the crystallization mushroom. The results show that (i) the heterogenenous nucleation does not crucially affect the nucleation process under conditions of high supersaturation and that (ii) the nucleating action of the fianctionalized surfaces is mainly related to their superficial charge distribution more than to their absolute charge, according to both classical and colloidal nucleation theories.
Acta Crystallographica Section F Structural Biology and Crystallization Communications, 2012
Pseudomonas putidaPtxS is a member of the LacI protein family of transcriptional regulators invol... more Pseudomonas putidaPtxS is a member of the LacI protein family of transcriptional regulators involved in glucose metabolism. All genes involved in this pathway are clustered into two operons,kguandgad. PtxS controls the expression of thekguandgadoperons as well as its own transcription. The PtxS operator is a perfect palindrome, 5′-TGAAACCGGTTTCA-3′, which is present in all three promoters. Crystallization of native PtxS failed, and PtxS–DNA crystals were finally produced by the counter-diffusion technique. A portion of the capillary used for crystal growth was attached to the end of a SPINE standard cap and directly flash-cooled in liquid nitrogen for diffraction tests. A full data set was collected with a beam size of 10 × 10 µm. The crystal belonged to the trigonal space groupP3, with unit-cell parametersa=b= 213.71,c = 71.57 Å. Only unhandled crystals grown in capillaries of 0.1 mm inner diameter diffracted X-rays to 1.92 Å resolution.
Acta Crystallographica Section D Biological Crystallography, 2011
SH3 domains are small protein modules that mediate the assembly of specific protein complexes, ty... more SH3 domains are small protein modules that mediate the assembly of specific protein complexes, typically via binding to proline-rich sequences in their respective binding partners. Most of the α-spectrin SH3-domain (Spc-SH3) structures determined to date using X-ray diffraction have been solved from crystals belonging to the orthorhombic space group P2(1)2(1)2(1) with a needle-like morphology. All of these orthorhombic crystals exhibited a rapid growth rate. In addition to this crystal form, the R21D mutant of Spc-SH3 crystallizes in a new crystal form in the presence of sodium formate at pH values higher than 6. This new crystal form grows at a slower rate and belongs to the hexagonal space group P6(5)22, with unit-cell parameters a = b = 42.9, c = 127.5 Å. When both polymorphs of the R21D mutant of Spc-SH3 are simultaneously present into the same solution, it has been observed that the hexagonal crystals grow at the expense of the orthorhombic crystals. The availability of 1.1 Å resolution structures for both crystal forms allows the identification of key features that could account for the observed polymorphic behaviour.
Acta Crystallographica Section A Foundations of Crystallography, 2004
Acta Crystallographica Section A Foundations of Crystallography, 2004
Journal of Crystal Growth, 2001
ABSTRACT
International Journal of Biological Macromolecules