Eric Baldwin - Academia.edu (original) (raw)
Papers by Eric Baldwin
Acta Crystallographica Section D Biological Crystallography, 2002
In bacteria the biosynthesis of all nascent polypeptides begins with Nformylmethionine. The post-... more In bacteria the biosynthesis of all nascent polypeptides begins with Nformylmethionine. The post-translational removal of the N-formyl group is carried out by peptide deformylase (PDF). Processing of the N-formyl group from critical bacterial proteins is required for cell survival. This formylation/deformylation cycle is unique to eubacteria and is not utilized in eucaryotic cytosolic protein biosynthesis. Thus, inhibition of PDF would halt bacterial growth, spare host cellfunction, and would be a novel mechanism for a new class of antibiotic. Diffraction-quality Se-met crystals of S. aureus PDF were prepared that belong to space group C222 1 with unit cell parameters of a = 94.1 b = 121.9 c = 47.6 A Ê. Multiple anomalous dispersion data were collected at the Advanced Photon Source 17-ID beamline and used to solve the PDF structure to 1.9 A Ê resolution. Crystals were also prepared with three PDF inhibitors: thiorphan, actinonin and PNU-172550. The thiorphan and actinonin co-crystals belong to space group C222 1 with similar unit-cell dimensions. Repeated attempts to generate a complex structure of PDF with PNU-172550 from the orthorhombic space group were unsuccessful. Crystallization screening identi®ed an alternate C2 crystal form with unitcell dimensions of a = 93.4 b = 42.5 c = 104.1 A Ê , = 93 .
L'invention concerne des anticorps isoles qui se lient de maniere immunospecifique a ROR1, de... more L'invention concerne des anticorps isoles qui se lient de maniere immunospecifique a ROR1, des anticorps bispecifiques comprenant un site de liaison a l'antigene qui se lie de maniere immunospecifique a ROR1 et un site de liaison a l'antigene qui se lie de maniere immunospecifique a CD3, et leurs procedes d'utilisation.
Blood Advances
B-cell maturation antigen (BCMA), a member of the tumor necrosis factor family of receptors, is p... more B-cell maturation antigen (BCMA), a member of the tumor necrosis factor family of receptors, is predominantly expressed on the surface of terminally differentiated B cells. BCMA is highly expressed on plasmablasts and plasma cells from multiple myeloma (MM) patient samples. We developed a BCMAxCD3 bispecific antibody (teclistamab [JNJ-64007957]) to recruit and activate T cells to kill BCMA-expressing MM cells. Teclistamab induced cytotoxicity of BCMA+ MM cell lines in vitro (H929 cells, 50% effective concentration [EC50] = 0.15 nM; MM.1R cells, EC50 = 0.06 nM; RPMI 8226 cells, EC50 = 0.45 nM) with concomitant T-cell activation (H929 cells, EC50 = 0.21 nM; MM.1R cells, EC50 = 0.1 nM; RPMI 8226 cells, EC50 = 0.28 nM) and cytokine release. This activity was further increased in the presence of a γ-secretase inhibitor (LY-411575). Teclistamab also depleted BCMA+ cells in bone marrow samples from MM patients in an ex vivo assay with an average EC50 value of 1.7 nM. Under more physiologic...
Blood
JNJ-64007957 is a bispecific antibody that binds to CD3 on T cells, and BCMA on plasma cells, and... more JNJ-64007957 is a bispecific antibody that binds to CD3 on T cells, and BCMA on plasma cells, and should induce T cell mediated killing of BCMA expressing malignant plasma cells. The objectives of this study were to characterize the tolerability of JNJ-64007957 when given intravenously as either single- or repeated-doses (5 total doses) to male cynomolgus monkeys. Pharmacokinetics (PK) and pharmacodynamics (PD) were evaluated in the repeat-dose groups; the single dose arms allowed for PK evaluations through Day 56, and determination of key PK parameters to support FIH dose modeling. Methods: The cynomolgus monkey was chosen for this study as JNJ-64007957 binds to both cynomolgus monkey CD3 and BCMA and it is an accepted non-rodent species for nonclinical tolerability, PK and PD evaluations. In this study, male monkeys (3/group) were administered either control or JNJ-64007957 via slow intravenous bolus injection on Days 1, 8, 15, 22, and 29 for repeat dose groups (1-4) and on Day 1 ...
Blood
Redirecting T cells to specifically target and kill malignant cells has been validated as an effe... more Redirecting T cells to specifically target and kill malignant cells has been validated as an effective anti-cancer strategy in the clinic with the approval of CD19xCD3 BiTE (Blincyto) for acute lymphoblastic leukemia. However, the immunosuppressive nature of the tumor microenvironment potentially poses a significant hurdle to T cell therapies. For instance, the bone marrow (BM) niche is appreciated to be a site of immune privilege at steady state to allow for normal hematopoiesis and immune cell generation. Additionally, in hematological malignancies, the BM niche is protective to leukemic stem cells, a phenomenon that has minimized the efficacy of several anti-cancer drugs including chemotherapy, targeted small molecule inhibitors and antibody based therapies. In this study, we investigated the impact of the BM microenvironment on T cell redirection. Using antibodies made with the Genmab DuoBody® technology targeting specific tumor antigens (CD123 and BCMA) and CD3, we observed tha...
Blood
B-cell maturation antigen (BCMA) is a tumor necrosis factor (TNF) family surface protein predomin... more B-cell maturation antigen (BCMA) is a tumor necrosis factor (TNF) family surface protein predominantly expressed on terminally differentiated B-cells. BCMA signals through P38/NF-κB pathway upon binding to its ligands; a proliferation inducing ligand (APRIL) and B-cell activator of the TNF family (BAFF) and promote anti-apoptotic gene expression. BCMA expression is elevated in plasma blasts, plasma cells from spleen and bone marrow and correlates with disease progression in multiple myeloma (MM). BCMA expression in premalignant MM settings such as monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM) also gives an opportunity for early cancer interception. To target cancer cells expressing BCMA, we developed a BCMAxCD3 bispecific antibody using the Genmab DuoBody® technology (Ab-957) to recruit T cells to BCMA-expressing MM cells so that T cells could be activated and induced to kill BCMA+ cancer cells. This antibody can induce cytotoxicity ...
Analytical Biochemistry, Oct 15, 2002
Several small molecules identified by high-throughput screening (HTS) were evaluated for their ab... more Several small molecules identified by high-throughput screening (HTS) were evaluated for their ability to bind to a nonstructural protein 3 (NS3) helicase from hepatitis C virus (HCV). Equilibrium dissociation constants (K d 's) of the compounds for this helicase were determined using several techniques including an assay measuring the kinetics of isothermal enzyme denaturation at several concentrations of the test molecule. Effects of two nonhydrolyzable ATP analogs on helicase denaturation were measured as controls using the isothermal denaturation (ITD) assay. Two compounds, 4-(2,4-dimethylphenyl)-2,7,8-trimethyl-4,5-quinolinediamine and 2-phenyl-N-(5-piperazin-1-ylpentyl)quinazolin-4-amine, were identified from screening that inhibited the enzyme and had low micromolar dissociation constants for NS3 helicase in the ITD assay. Low micromolar affinity of the quinolinediamine to helicase was also confirmed by nuclear magnetic resonance experiments. Unfortunately, isothermal titration calorimetry (ITC) experiments indicated that a more water-soluble analog bound to the 47/23-mer oligonucleotide helicase substrate with low micromolar affinity as did the substituted quinazolinamine. There was no further interest in these templates as helicase inhibitors due to the nonspecific binding to enzyme and substrate. A combination of physical methods was required to discern the mode of action of compounds identified by HTS and remove undesirable lead templates from further consideration.
Genome Medicine, 2015
Background: In an effort to return actionable results from variant data to electronic health reco... more Background: In an effort to return actionable results from variant data to electronic health records (EHRs), participants in the Electronic Medical Records and Genomics (eMERGE) Network are being sequenced with the targeted Pharmacogenomics Research Network sequence platform (PGRNseq). This cost-effective, highly-scalable, and highly-accurate platform was created to explore rare variation in 84 key pharmacogenetic genes with strong drug phenotype associations. Methods: To return Clinical Laboratory Improvement Amendments (CLIA) results to our participants at the Group Health Cooperative, we sequenced the DNA of 900 participants (61 % female) with non-CLIA biobanked samples. We then selected 450 of those to be re-consented, to redraw blood, and ultimately to validate CLIA variants in anticipation of returning the results to the participant and EHR. These 450 were selected using an algorithm we designed to harness data from self-reported race, diagnosis and procedure codes, medical notes, laboratory results, and variant-level bioinformatics to ensure selection of an informative sample. We annotated the multi-sample variant call format by a combination of SeattleSeq and SnpEff tools, with additional custom variables including evidence from ClinVar, OMIM, HGMD, and prior clinical associations. Results: We focused our analyses on 27 actionable genes, largely driven by the Clinical Pharmacogenetics Implementation Consortium. We derived a ranking system based on the total number of coding variants per participant (75.2 ± 14.7), and the number of coding variants with high or moderate impact (11.5 ± 3.9). Notably, we identified 11 stop-gained (1 %) and 519 missense (20 %) variants out of a total of 1785 in these 27 genes. Finally, we prioritized variants to be returned to the EHR with prior clinical evidence of pathogenicity or annotated as stop-gain for the following genes: CACNA1S and RYR1 (malignant hyperthermia); SCN5A, KCNH2, and RYR2 (arrhythmia); and LDLR (high cholesterol). Conclusions: The incorporation of genetics into the EHR for clinical decision support is a complex undertaking for many reasons including lack of prior consent for return of results, lack of biospecimens collected in a CLIA environment, and EHR integration. Our study design accounts for these hurdles and is an example of a pilot system that can be utilized before expanding to an entire health system.
Bioorganic & Medicinal Chemistry Letters, 2015
Glycogen synthase kinase-3 (GSK-3) has been proposed to play a crucial role in the pathogenesis o... more Glycogen synthase kinase-3 (GSK-3) has been proposed to play a crucial role in the pathogenesis of many diseases including cancer, stroke, bipolar disorders, diabetes and neurodegenerative diseases. GSK-3 inhibition has been a major area of pharmaceutical interest over the last two decades. A plethora of reports appeared recently on selective inhibitors and their co-crystal structures in GSK-3β. We identified several series of promising new GSK-3β inhibitors from a coherent design around a pyrrolopyridinone core structure. A systematic exploration of the chemical space around the central spacer led to potent single digit and sub-nanomolar GSK-3β inhibitors. When dosed orally in a transgenic mouse model of Alzheimer's disease (AD), an exemplary compound showed significant lowering of Tau phosphorylation at one of the GSK-3 phosphorylating sites, Ser396. X-ray crystallography greatly aided in validating the binding hypotheses.
Nature structural biology, 1994
HIV-1 proteinase (HIV PR) is a dimeric enzyme composed of two identical polypeptide chains that a... more HIV-1 proteinase (HIV PR) is a dimeric enzyme composed of two identical polypeptide chains that associate with twofold symmetry. We have determined to 1.8 A the crystal structure of a covalently tethered dimer of HIV PR. The tethered dimer:inhibitor complex is identical in nearly every respect to the complex of the same inhibitor with the wild type dimeric molecule, except for the linker region. Our results suggest that the tethered dimer may be a useful surrogate enzyme for studying the effects of single site mutations on substrate and inhibitor binding as well as on enzyme asymmetry, and for simulating independent mutational drift of the two domains which has been proposed to have led to the evolution of modern day, single-chain aspartic proteinases.
Advances in Experimental Medicine and Biology, 1995
Either your web browser doesn't support Javascript or it is currently turned off. In the... more Either your web browser doesn't support Javascript or it is currently turned off. In the latter case, please turn on Javascript support in your web browser and reload this page. ... Structure of HIV-1 protease with KNI-272: a transition state mimetic inhibitor containing ...
Background: The integrin family of cell-surface receptors mediate cell adhesion through interacti... more Background: The integrin family of cell-surface receptors mediate cell adhesion through interactions with the extracellular matrix or other cell-surface receptors. The α chain of some integrin heterodimers includes an inserted 'I domain' of about 200 amino acids which binds divalent metal ions and is essential for integrin function. Lee et al. proposed that the I domain of the integrin CD11b adopts a unique 'active' conformation when bound to its counter receptor. In addition, they proposed that the lack of adhesion in the presence of Ca 2+ ion reflected the stabilization of an 'inactive' I-domain conformation. We set out to independently determine the structure of the CD11b I domain and to evaluate the structural effects of divalent ion binding to this protein. Results: We have determined the X-ray structure of a new crystal form of the CD11b I domain in the absence of added metal ions by multiple isomorphous replacement (MIR). Metal ions were easily introduced into this crystal form allowing the straightforward assessment of the structural effects of divalent cation binding at the metal ion dependent adhesion site (MIDAS). The equilibrium binding constants for these ions were determined by titration calorimetry. The overall protein conformation and metal-ion coordination of the I domain is the same as that observed for all previously reported CD11a I-domain structures and a CD11b I-domain complex with Mn 2+. These structures define a majority conformation. Conclusions: Addition of the cations Mg 2+ , Mn 2+ and Cd 2+ to the metal-free I domain does not induce conformational changes in the crystalline environment. Moreover, we find that Ca 2+ binds poorly to the I domain which serves to explain its failure to support adhesion. We show that the active conformation proposed by Lee et al. is likely to be a construct artifact and we propose that the currently available data do not support a dramatic structural transition for the I domain during counter-receptor binding.
Advances in Experimental Medicine and Biology, 1995
1. Adv Exp Med Biol. 1995;362:181-92. Structure of human cathepsin D: comparison of inhibitor bin... more 1. Adv Exp Med Biol. 1995;362:181-92. Structure of human cathepsin D: comparison of inhibitor binding and subdomain displacement with other aspartic proteases. Erickson JW, Baldwin ET, Bhat TN, Gulnik S. Structural Biochemistry ...
Chemical Immunology and Allergy, 1992
Acta Crystallographica Section D Biological Crystallography, 2002
In bacteria the biosynthesis of all nascent polypeptides begins with Nformylmethionine. The post-... more In bacteria the biosynthesis of all nascent polypeptides begins with Nformylmethionine. The post-translational removal of the N-formyl group is carried out by peptide deformylase (PDF). Processing of the N-formyl group from critical bacterial proteins is required for cell survival. This formylation/deformylation cycle is unique to eubacteria and is not utilized in eucaryotic cytosolic protein biosynthesis. Thus, inhibition of PDF would halt bacterial growth, spare host cellfunction, and would be a novel mechanism for a new class of antibiotic. Diffraction-quality Se-met crystals of S. aureus PDF were prepared that belong to space group C222 1 with unit cell parameters of a = 94.1 b = 121.9 c = 47.6 A Ê. Multiple anomalous dispersion data were collected at the Advanced Photon Source 17-ID beamline and used to solve the PDF structure to 1.9 A Ê resolution. Crystals were also prepared with three PDF inhibitors: thiorphan, actinonin and PNU-172550. The thiorphan and actinonin co-crystals belong to space group C222 1 with similar unit-cell dimensions. Repeated attempts to generate a complex structure of PDF with PNU-172550 from the orthorhombic space group were unsuccessful. Crystallization screening identi®ed an alternate C2 crystal form with unitcell dimensions of a = 93.4 b = 42.5 c = 104.1 A Ê , = 93 .
L'invention concerne des anticorps isoles qui se lient de maniere immunospecifique a ROR1, de... more L'invention concerne des anticorps isoles qui se lient de maniere immunospecifique a ROR1, des anticorps bispecifiques comprenant un site de liaison a l'antigene qui se lie de maniere immunospecifique a ROR1 et un site de liaison a l'antigene qui se lie de maniere immunospecifique a CD3, et leurs procedes d'utilisation.
Blood Advances
B-cell maturation antigen (BCMA), a member of the tumor necrosis factor family of receptors, is p... more B-cell maturation antigen (BCMA), a member of the tumor necrosis factor family of receptors, is predominantly expressed on the surface of terminally differentiated B cells. BCMA is highly expressed on plasmablasts and plasma cells from multiple myeloma (MM) patient samples. We developed a BCMAxCD3 bispecific antibody (teclistamab [JNJ-64007957]) to recruit and activate T cells to kill BCMA-expressing MM cells. Teclistamab induced cytotoxicity of BCMA+ MM cell lines in vitro (H929 cells, 50% effective concentration [EC50] = 0.15 nM; MM.1R cells, EC50 = 0.06 nM; RPMI 8226 cells, EC50 = 0.45 nM) with concomitant T-cell activation (H929 cells, EC50 = 0.21 nM; MM.1R cells, EC50 = 0.1 nM; RPMI 8226 cells, EC50 = 0.28 nM) and cytokine release. This activity was further increased in the presence of a γ-secretase inhibitor (LY-411575). Teclistamab also depleted BCMA+ cells in bone marrow samples from MM patients in an ex vivo assay with an average EC50 value of 1.7 nM. Under more physiologic...
Blood
JNJ-64007957 is a bispecific antibody that binds to CD3 on T cells, and BCMA on plasma cells, and... more JNJ-64007957 is a bispecific antibody that binds to CD3 on T cells, and BCMA on plasma cells, and should induce T cell mediated killing of BCMA expressing malignant plasma cells. The objectives of this study were to characterize the tolerability of JNJ-64007957 when given intravenously as either single- or repeated-doses (5 total doses) to male cynomolgus monkeys. Pharmacokinetics (PK) and pharmacodynamics (PD) were evaluated in the repeat-dose groups; the single dose arms allowed for PK evaluations through Day 56, and determination of key PK parameters to support FIH dose modeling. Methods: The cynomolgus monkey was chosen for this study as JNJ-64007957 binds to both cynomolgus monkey CD3 and BCMA and it is an accepted non-rodent species for nonclinical tolerability, PK and PD evaluations. In this study, male monkeys (3/group) were administered either control or JNJ-64007957 via slow intravenous bolus injection on Days 1, 8, 15, 22, and 29 for repeat dose groups (1-4) and on Day 1 ...
Blood
Redirecting T cells to specifically target and kill malignant cells has been validated as an effe... more Redirecting T cells to specifically target and kill malignant cells has been validated as an effective anti-cancer strategy in the clinic with the approval of CD19xCD3 BiTE (Blincyto) for acute lymphoblastic leukemia. However, the immunosuppressive nature of the tumor microenvironment potentially poses a significant hurdle to T cell therapies. For instance, the bone marrow (BM) niche is appreciated to be a site of immune privilege at steady state to allow for normal hematopoiesis and immune cell generation. Additionally, in hematological malignancies, the BM niche is protective to leukemic stem cells, a phenomenon that has minimized the efficacy of several anti-cancer drugs including chemotherapy, targeted small molecule inhibitors and antibody based therapies. In this study, we investigated the impact of the BM microenvironment on T cell redirection. Using antibodies made with the Genmab DuoBody® technology targeting specific tumor antigens (CD123 and BCMA) and CD3, we observed tha...
Blood
B-cell maturation antigen (BCMA) is a tumor necrosis factor (TNF) family surface protein predomin... more B-cell maturation antigen (BCMA) is a tumor necrosis factor (TNF) family surface protein predominantly expressed on terminally differentiated B-cells. BCMA signals through P38/NF-κB pathway upon binding to its ligands; a proliferation inducing ligand (APRIL) and B-cell activator of the TNF family (BAFF) and promote anti-apoptotic gene expression. BCMA expression is elevated in plasma blasts, plasma cells from spleen and bone marrow and correlates with disease progression in multiple myeloma (MM). BCMA expression in premalignant MM settings such as monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM) also gives an opportunity for early cancer interception. To target cancer cells expressing BCMA, we developed a BCMAxCD3 bispecific antibody using the Genmab DuoBody® technology (Ab-957) to recruit T cells to BCMA-expressing MM cells so that T cells could be activated and induced to kill BCMA+ cancer cells. This antibody can induce cytotoxicity ...
Analytical Biochemistry, Oct 15, 2002
Several small molecules identified by high-throughput screening (HTS) were evaluated for their ab... more Several small molecules identified by high-throughput screening (HTS) were evaluated for their ability to bind to a nonstructural protein 3 (NS3) helicase from hepatitis C virus (HCV). Equilibrium dissociation constants (K d 's) of the compounds for this helicase were determined using several techniques including an assay measuring the kinetics of isothermal enzyme denaturation at several concentrations of the test molecule. Effects of two nonhydrolyzable ATP analogs on helicase denaturation were measured as controls using the isothermal denaturation (ITD) assay. Two compounds, 4-(2,4-dimethylphenyl)-2,7,8-trimethyl-4,5-quinolinediamine and 2-phenyl-N-(5-piperazin-1-ylpentyl)quinazolin-4-amine, were identified from screening that inhibited the enzyme and had low micromolar dissociation constants for NS3 helicase in the ITD assay. Low micromolar affinity of the quinolinediamine to helicase was also confirmed by nuclear magnetic resonance experiments. Unfortunately, isothermal titration calorimetry (ITC) experiments indicated that a more water-soluble analog bound to the 47/23-mer oligonucleotide helicase substrate with low micromolar affinity as did the substituted quinazolinamine. There was no further interest in these templates as helicase inhibitors due to the nonspecific binding to enzyme and substrate. A combination of physical methods was required to discern the mode of action of compounds identified by HTS and remove undesirable lead templates from further consideration.
Genome Medicine, 2015
Background: In an effort to return actionable results from variant data to electronic health reco... more Background: In an effort to return actionable results from variant data to electronic health records (EHRs), participants in the Electronic Medical Records and Genomics (eMERGE) Network are being sequenced with the targeted Pharmacogenomics Research Network sequence platform (PGRNseq). This cost-effective, highly-scalable, and highly-accurate platform was created to explore rare variation in 84 key pharmacogenetic genes with strong drug phenotype associations. Methods: To return Clinical Laboratory Improvement Amendments (CLIA) results to our participants at the Group Health Cooperative, we sequenced the DNA of 900 participants (61 % female) with non-CLIA biobanked samples. We then selected 450 of those to be re-consented, to redraw blood, and ultimately to validate CLIA variants in anticipation of returning the results to the participant and EHR. These 450 were selected using an algorithm we designed to harness data from self-reported race, diagnosis and procedure codes, medical notes, laboratory results, and variant-level bioinformatics to ensure selection of an informative sample. We annotated the multi-sample variant call format by a combination of SeattleSeq and SnpEff tools, with additional custom variables including evidence from ClinVar, OMIM, HGMD, and prior clinical associations. Results: We focused our analyses on 27 actionable genes, largely driven by the Clinical Pharmacogenetics Implementation Consortium. We derived a ranking system based on the total number of coding variants per participant (75.2 ± 14.7), and the number of coding variants with high or moderate impact (11.5 ± 3.9). Notably, we identified 11 stop-gained (1 %) and 519 missense (20 %) variants out of a total of 1785 in these 27 genes. Finally, we prioritized variants to be returned to the EHR with prior clinical evidence of pathogenicity or annotated as stop-gain for the following genes: CACNA1S and RYR1 (malignant hyperthermia); SCN5A, KCNH2, and RYR2 (arrhythmia); and LDLR (high cholesterol). Conclusions: The incorporation of genetics into the EHR for clinical decision support is a complex undertaking for many reasons including lack of prior consent for return of results, lack of biospecimens collected in a CLIA environment, and EHR integration. Our study design accounts for these hurdles and is an example of a pilot system that can be utilized before expanding to an entire health system.
Bioorganic & Medicinal Chemistry Letters, 2015
Glycogen synthase kinase-3 (GSK-3) has been proposed to play a crucial role in the pathogenesis o... more Glycogen synthase kinase-3 (GSK-3) has been proposed to play a crucial role in the pathogenesis of many diseases including cancer, stroke, bipolar disorders, diabetes and neurodegenerative diseases. GSK-3 inhibition has been a major area of pharmaceutical interest over the last two decades. A plethora of reports appeared recently on selective inhibitors and their co-crystal structures in GSK-3β. We identified several series of promising new GSK-3β inhibitors from a coherent design around a pyrrolopyridinone core structure. A systematic exploration of the chemical space around the central spacer led to potent single digit and sub-nanomolar GSK-3β inhibitors. When dosed orally in a transgenic mouse model of Alzheimer's disease (AD), an exemplary compound showed significant lowering of Tau phosphorylation at one of the GSK-3 phosphorylating sites, Ser396. X-ray crystallography greatly aided in validating the binding hypotheses.
Nature structural biology, 1994
HIV-1 proteinase (HIV PR) is a dimeric enzyme composed of two identical polypeptide chains that a... more HIV-1 proteinase (HIV PR) is a dimeric enzyme composed of two identical polypeptide chains that associate with twofold symmetry. We have determined to 1.8 A the crystal structure of a covalently tethered dimer of HIV PR. The tethered dimer:inhibitor complex is identical in nearly every respect to the complex of the same inhibitor with the wild type dimeric molecule, except for the linker region. Our results suggest that the tethered dimer may be a useful surrogate enzyme for studying the effects of single site mutations on substrate and inhibitor binding as well as on enzyme asymmetry, and for simulating independent mutational drift of the two domains which has been proposed to have led to the evolution of modern day, single-chain aspartic proteinases.
Advances in Experimental Medicine and Biology, 1995
Either your web browser doesn't support Javascript or it is currently turned off. In the... more Either your web browser doesn't support Javascript or it is currently turned off. In the latter case, please turn on Javascript support in your web browser and reload this page. ... Structure of HIV-1 protease with KNI-272: a transition state mimetic inhibitor containing ...
Background: The integrin family of cell-surface receptors mediate cell adhesion through interacti... more Background: The integrin family of cell-surface receptors mediate cell adhesion through interactions with the extracellular matrix or other cell-surface receptors. The α chain of some integrin heterodimers includes an inserted 'I domain' of about 200 amino acids which binds divalent metal ions and is essential for integrin function. Lee et al. proposed that the I domain of the integrin CD11b adopts a unique 'active' conformation when bound to its counter receptor. In addition, they proposed that the lack of adhesion in the presence of Ca 2+ ion reflected the stabilization of an 'inactive' I-domain conformation. We set out to independently determine the structure of the CD11b I domain and to evaluate the structural effects of divalent ion binding to this protein. Results: We have determined the X-ray structure of a new crystal form of the CD11b I domain in the absence of added metal ions by multiple isomorphous replacement (MIR). Metal ions were easily introduced into this crystal form allowing the straightforward assessment of the structural effects of divalent cation binding at the metal ion dependent adhesion site (MIDAS). The equilibrium binding constants for these ions were determined by titration calorimetry. The overall protein conformation and metal-ion coordination of the I domain is the same as that observed for all previously reported CD11a I-domain structures and a CD11b I-domain complex with Mn 2+. These structures define a majority conformation. Conclusions: Addition of the cations Mg 2+ , Mn 2+ and Cd 2+ to the metal-free I domain does not induce conformational changes in the crystalline environment. Moreover, we find that Ca 2+ binds poorly to the I domain which serves to explain its failure to support adhesion. We show that the active conformation proposed by Lee et al. is likely to be a construct artifact and we propose that the currently available data do not support a dramatic structural transition for the I domain during counter-receptor binding.
Advances in Experimental Medicine and Biology, 1995
1. Adv Exp Med Biol. 1995;362:181-92. Structure of human cathepsin D: comparison of inhibitor bin... more 1. Adv Exp Med Biol. 1995;362:181-92. Structure of human cathepsin D: comparison of inhibitor binding and subdomain displacement with other aspartic proteases. Erickson JW, Baldwin ET, Bhat TN, Gulnik S. Structural Biochemistry ...
Chemical Immunology and Allergy, 1992