The molecular weight-gyration radius relation for a number of globular proteins based on experime... more The molecular weight-gyration radius relation for a number of globular proteins based on experimental light scattering data is compared with small-angle X-ray scattering data recently published by Mylonas & Svergun [J. Appl. Cryst. (2007 ▸), 40, s245-s249]. In addition, other recent experimental data and theoretical calculations are reviewed. It is found that the M W-R g relation for the globular proteins is well represented by a power law with an exponent of 0.37 (2).
Diseases of ectopic calcification of the vascular wall range from lethal orphan diseases such as ... more Diseases of ectopic calcification of the vascular wall range from lethal orphan diseases such as generalized arterial calcification of infancy (GACI), to common diseases such as hardening of the arteries associated with aging and calciphylaxis of chronic kidney disease (CKD). GACI is a lethal orphan disease in which infants calcify the internal elastic lamina of their medium and large arteries and expire of cardiac failure as neonates, while calciphylaxis of CKD is a ubiquitous vascular calcification in patients with renal failure. Both disorders are characterized by vascular Mönckeburg's sclerosis accompanied by decreased concentrations of plasma inorganic pyrophosphate (PPi). Here we demonstrate that subcutaneous administration of an ENPP1-Fc fusion protein prevents the mortality, vascular calcifications and sequela of disease in animal models of GACI, and is accompanied by a complete clinical and biomarker response. Our findings have implications for the treatment of rare and common diseases of ectopic vascular calcification.
The receptor tyrosine kinase KIT plays an important role in development of germ cells, hematopoie... more The receptor tyrosine kinase KIT plays an important role in development of germ cells, hematopoietic cells, and interstitial pacemaker cells. Oncogenic KIT mutations play an important "driver" role in gastrointestinal stromal tumors, acute myeloid leukemias, and melanoma, among other cancers. Here we describe the crystal structure of a recurring somatic oncogenic mutation located in the C-terminal Ig-like domain (D5) of the ectodomain, rendering KIT tyrosine kinase activity constitutively activated. The structural analysis, together with biochemical and biophysical experiments and detailed analyses of the activities of a variety of oncogenic KIT mutations, reveals that the strength of homotypic contacts and the cooperativity in the action of D4D5 regions determines whether KIT is normally regulated or constitutively activated in cancers. We propose that cooperative interactions mediated by multiple weak homotypic contacts between receptor molecules are responsible for regu...
Methods in molecular biology (Clifton, N.J.), 2006
Size-exclusion chromatography (SEC), coupled with "on-line" static laser light scatteri... more Size-exclusion chromatography (SEC), coupled with "on-line" static laser light scattering (LS), refractive index (RI), and ultraviolet (UV) detection, provides a universal approach for determination of the molar mass and oligomeric state in solution of native proteins as well as glycosylated proteins or membrane proteins solubilized in non-ionic detergents. Such glycosylated proteins or protein-detergent complexes show anomalous behavior on SEC, thus presenting a challenge to determination of molar mass and oligomeric state in solution. In the SEC-UV/LS/RI approach, SEC serves solely as a fractionation step, while the responses from the three detectors are utilized to calculate the molar mass for the polypeptide portion of the native or modified protein. The amount of sugar, lipid, or detergent bound to the polypeptide chain can also be estimated from the SEC-UV/LS/RI analysis.
Anaplastic lymphoma kinase (ALK) is one of the few remaining "orphan" receptor tyrosine... more Anaplastic lymphoma kinase (ALK) is one of the few remaining "orphan" receptor tyrosine kinases (RTKs) in which the ligands are unknown. Ligand-mediated activation of RTKs is important throughout development. ALK is particularly relevant to the development of the nervous system. Increased activation of RTKs by mutation, genetic amplification, or signals from the stroma contributes to disease progression and acquired drug resistance in cancer. Aberrant activation of ALK occurs in subsets of lung adenocarcinoma, neuroblastoma, and other cancers. We found that heparin is a ligand that binds specifically to the ALK extracellular domain. Whereas heparins with short chain lengths bound to ALK in a monovalent manner and did not activate the receptor, longer heparin chains induced ALK dimerization and activation in cultured neuroblastoma cells. Heparin lacking N- and O-linked sulfate groups or other glycosaminoglycans with sulfation patterns different than heparin failed to activa...
Cerebral cavernous malformation (CCM) is a disease that affects between 0.1 and 0.5% of the human... more Cerebral cavernous malformation (CCM) is a disease that affects between 0.1 and 0.5% of the human population, with mutations in CCM3 accounting for ~ 15% of the autosomal dominant form of the disease. We recently reported that CCM3 contains an N-terminal dimerization domain (CCM3D) and a C-terminal focal adhesion targeting (FAT) homology domain. Intermolecular protein-protein interactions of CCM3 are mediated by a highly conserved surface on the FAT homology domain and are affected by CCM3 truncations in the human disease. Here we report the crystal structures of CCM3 in complex with three different leucine-aspartate repeat (LD) motifs (LD1, LD2, and LD4) from the scaffolding protein paxillin, at 2.8, 2.7, and 2.5 Å resolution. We show that CCM3 binds LD motifs using the highly conserved hydrophobic patch 1 (HP1) and that this binding is similar to the binding of focal adhesion kinase and Pyk2 FAT domains to paxillin LD motifs. We further show by surface plasmon resonance that CCM3 binds paxillin LD motifs with affinities in the micromolar range, similar to FAK family FAT domains. Finally, we show that endogenous CCM3 and paxillin co-localize in mouse cerebral pericytes. These studies provide a molecular-level framework to investigate the protein-protein interactions of CCM3.
The SecA nanomotor promotes protein translocation in eubacteria by binding both protein cargo and... more The SecA nanomotor promotes protein translocation in eubacteria by binding both protein cargo and the protein-conducting channel and by undergoing ATP-driven conformation cycles that drive this process. There are conflicting reports about whether SecA functions as a ...
Characterization of the kinetics and energetics of base-pair opening in nucleic acids relies upon... more Characterization of the kinetics and energetics of base-pair opening in nucleic acids relies upon measurements of the rates of exchange of imino protons with water protons at high concentrations of the exchange catalyst. Under these conditions, the exchange catalyst may affect structural or dynamic properties of the nucleic acid molecule and thus, limit the significance of the exchange data. To address this problem, we have used NMR spectroscopy to characterize the effects of a catalyst of imino proton exchange, namely, ammonia upon the structure and dynamics of the self-complementary DNA dodecamer [d(CGCAGATCTGCG)]2. The changes in structure were monitored in proton NOESY and DQF-COSY experiments and in phosphorus spectra at 15 degrees C and at ammonia concentrations ranging from 0.002 to 0.5 M. The results indicate that ammonia induces subtle changes in the solution conformation of the dodecamer, but the overall structure is maintained close to the B-type DNA structure. However, the relaxation rates (i.e., transverse, longitudinal, and cross-relaxation rates) of several non-exchangeable protons were found to increase by approximately 50% upon changing ammonia concentration from 0.002 to 0.5 M. The increases were comparable for all protons investigated suggesting that they originate from an ammonia-induced increase in the overall correlation time of the DNA dodecamer. Numerical analysis revealed that the catalyst-induced enhancements in proton relaxation can alter significantly the calculated values of the exchange rates of imino protons, especially those obtained from measurements of the line widths of these proton resonances.
The molecular weight-gyration radius relation for a number of globular proteins based on experime... more The molecular weight-gyration radius relation for a number of globular proteins based on experimental light scattering data is compared with small-angle X-ray scattering data recently published by Mylonas & Svergun [J. Appl. Cryst. (2007 ▸), 40, s245-s249]. In addition, other recent experimental data and theoretical calculations are reviewed. It is found that the M W-R g relation for the globular proteins is well represented by a power law with an exponent of 0.37 (2).
Diseases of ectopic calcification of the vascular wall range from lethal orphan diseases such as ... more Diseases of ectopic calcification of the vascular wall range from lethal orphan diseases such as generalized arterial calcification of infancy (GACI), to common diseases such as hardening of the arteries associated with aging and calciphylaxis of chronic kidney disease (CKD). GACI is a lethal orphan disease in which infants calcify the internal elastic lamina of their medium and large arteries and expire of cardiac failure as neonates, while calciphylaxis of CKD is a ubiquitous vascular calcification in patients with renal failure. Both disorders are characterized by vascular Mönckeburg's sclerosis accompanied by decreased concentrations of plasma inorganic pyrophosphate (PPi). Here we demonstrate that subcutaneous administration of an ENPP1-Fc fusion protein prevents the mortality, vascular calcifications and sequela of disease in animal models of GACI, and is accompanied by a complete clinical and biomarker response. Our findings have implications for the treatment of rare and common diseases of ectopic vascular calcification.
The receptor tyrosine kinase KIT plays an important role in development of germ cells, hematopoie... more The receptor tyrosine kinase KIT plays an important role in development of germ cells, hematopoietic cells, and interstitial pacemaker cells. Oncogenic KIT mutations play an important "driver" role in gastrointestinal stromal tumors, acute myeloid leukemias, and melanoma, among other cancers. Here we describe the crystal structure of a recurring somatic oncogenic mutation located in the C-terminal Ig-like domain (D5) of the ectodomain, rendering KIT tyrosine kinase activity constitutively activated. The structural analysis, together with biochemical and biophysical experiments and detailed analyses of the activities of a variety of oncogenic KIT mutations, reveals that the strength of homotypic contacts and the cooperativity in the action of D4D5 regions determines whether KIT is normally regulated or constitutively activated in cancers. We propose that cooperative interactions mediated by multiple weak homotypic contacts between receptor molecules are responsible for regu...
Methods in molecular biology (Clifton, N.J.), 2006
Size-exclusion chromatography (SEC), coupled with "on-line" static laser light scatteri... more Size-exclusion chromatography (SEC), coupled with "on-line" static laser light scattering (LS), refractive index (RI), and ultraviolet (UV) detection, provides a universal approach for determination of the molar mass and oligomeric state in solution of native proteins as well as glycosylated proteins or membrane proteins solubilized in non-ionic detergents. Such glycosylated proteins or protein-detergent complexes show anomalous behavior on SEC, thus presenting a challenge to determination of molar mass and oligomeric state in solution. In the SEC-UV/LS/RI approach, SEC serves solely as a fractionation step, while the responses from the three detectors are utilized to calculate the molar mass for the polypeptide portion of the native or modified protein. The amount of sugar, lipid, or detergent bound to the polypeptide chain can also be estimated from the SEC-UV/LS/RI analysis.
Anaplastic lymphoma kinase (ALK) is one of the few remaining "orphan" receptor tyrosine... more Anaplastic lymphoma kinase (ALK) is one of the few remaining "orphan" receptor tyrosine kinases (RTKs) in which the ligands are unknown. Ligand-mediated activation of RTKs is important throughout development. ALK is particularly relevant to the development of the nervous system. Increased activation of RTKs by mutation, genetic amplification, or signals from the stroma contributes to disease progression and acquired drug resistance in cancer. Aberrant activation of ALK occurs in subsets of lung adenocarcinoma, neuroblastoma, and other cancers. We found that heparin is a ligand that binds specifically to the ALK extracellular domain. Whereas heparins with short chain lengths bound to ALK in a monovalent manner and did not activate the receptor, longer heparin chains induced ALK dimerization and activation in cultured neuroblastoma cells. Heparin lacking N- and O-linked sulfate groups or other glycosaminoglycans with sulfation patterns different than heparin failed to activa...
Cerebral cavernous malformation (CCM) is a disease that affects between 0.1 and 0.5% of the human... more Cerebral cavernous malformation (CCM) is a disease that affects between 0.1 and 0.5% of the human population, with mutations in CCM3 accounting for ~ 15% of the autosomal dominant form of the disease. We recently reported that CCM3 contains an N-terminal dimerization domain (CCM3D) and a C-terminal focal adhesion targeting (FAT) homology domain. Intermolecular protein-protein interactions of CCM3 are mediated by a highly conserved surface on the FAT homology domain and are affected by CCM3 truncations in the human disease. Here we report the crystal structures of CCM3 in complex with three different leucine-aspartate repeat (LD) motifs (LD1, LD2, and LD4) from the scaffolding protein paxillin, at 2.8, 2.7, and 2.5 Å resolution. We show that CCM3 binds LD motifs using the highly conserved hydrophobic patch 1 (HP1) and that this binding is similar to the binding of focal adhesion kinase and Pyk2 FAT domains to paxillin LD motifs. We further show by surface plasmon resonance that CCM3 binds paxillin LD motifs with affinities in the micromolar range, similar to FAK family FAT domains. Finally, we show that endogenous CCM3 and paxillin co-localize in mouse cerebral pericytes. These studies provide a molecular-level framework to investigate the protein-protein interactions of CCM3.
The SecA nanomotor promotes protein translocation in eubacteria by binding both protein cargo and... more The SecA nanomotor promotes protein translocation in eubacteria by binding both protein cargo and the protein-conducting channel and by undergoing ATP-driven conformation cycles that drive this process. There are conflicting reports about whether SecA functions as a ...
Characterization of the kinetics and energetics of base-pair opening in nucleic acids relies upon... more Characterization of the kinetics and energetics of base-pair opening in nucleic acids relies upon measurements of the rates of exchange of imino protons with water protons at high concentrations of the exchange catalyst. Under these conditions, the exchange catalyst may affect structural or dynamic properties of the nucleic acid molecule and thus, limit the significance of the exchange data. To address this problem, we have used NMR spectroscopy to characterize the effects of a catalyst of imino proton exchange, namely, ammonia upon the structure and dynamics of the self-complementary DNA dodecamer [d(CGCAGATCTGCG)]2. The changes in structure were monitored in proton NOESY and DQF-COSY experiments and in phosphorus spectra at 15 degrees C and at ammonia concentrations ranging from 0.002 to 0.5 M. The results indicate that ammonia induces subtle changes in the solution conformation of the dodecamer, but the overall structure is maintained close to the B-type DNA structure. However, the relaxation rates (i.e., transverse, longitudinal, and cross-relaxation rates) of several non-exchangeable protons were found to increase by approximately 50% upon changing ammonia concentration from 0.002 to 0.5 M. The increases were comparable for all protons investigated suggesting that they originate from an ammonia-induced increase in the overall correlation time of the DNA dodecamer. Numerical analysis revealed that the catalyst-induced enhancements in proton relaxation can alter significantly the calculated values of the exchange rates of imino protons, especially those obtained from measurements of the line widths of these proton resonances.
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