Nature structural & molecular biology, Jan 23, 2015
Immunoglobulin G4 antibodies exhibit unusual properties with important biological consequences. W... more Immunoglobulin G4 antibodies exhibit unusual properties with important biological consequences. We report the structure of the human full-length IgG4 S228P anti-PD1 antibody pembrolizumab, solved to 2.3-Å resolution. Pembrolizumab is a compact molecule, consistent with the presence of a short hinge region. The Fc domain is glycosylated at the CH2 domain on both chains, but one CH2 domain is rotated 120° with respect to the conformation observed in all reported structures to date, and its glycan chain faces the solvent. We speculate that this new conformation is driven by the shorter hinge. The structure suggests a role for the S228P mutation in preventing the IgG4 arm exchange. In addition, this unusual Fc conformation suggests possible structural diversity between IgG subclasses and shows that use of isolated antibody fragments could mask potentially important interactions, owing to molecular flexibility.
The presence of aggregates of Factor VIII (FVIII) was assessed for a highly purified form (rFVIII... more The presence of aggregates of Factor VIII (FVIII) was assessed for a highly purified form (rFVIII) and for the formulated preparation containing human serum albumin (rFVIIIf). Size-exclusion chromatography (SEC) on Sepharose CL-6B and TSK 4000 matrices under native conditions revealed < 1% aggregates of FVIII in either rFVIII or rFVIIIf, as determined by FVIII immunoassay of chromatographic fractions. No degradation products were observed. The immunoassay was capable of detecting FVIII immunoreactivity to levels of approximately 5 ng/ml. Overlap of FVIII clotting (FVIII:c) and immunoassay (FVIII:cAg) activities was observed for SEC fractions. Heat stressing of rFVIIIf (47.5 degrees C for 24 h) resulted in a quantitative increase in FVIII-positive material in the void volume of a TSK 4000 column, demonstrating that aggregates of FVIII can be produced and detected by this method. We conclude that aggregates of FVIII in rFVIII and rFVIIIf constitute < or = 1% by the method descri...
Aggregation of proteins is a major problem in their use as drugs and is also involved in a variet... more Aggregation of proteins is a major problem in their use as drugs and is also involved in a variety of pathological diseases. In this study, biophysical techniques were employed to investigate aggregate formation in the pharmaceutically important protein, recombinant human factor VIII (rhFVIII). Recombinant human factor VIII incubated in solution at 37 degrees C formed soluble aggregates as detected by molecular sieve chromatography and dynamic light scattering. This resulted in a corresponding loss of biological activity. Fluorescence and CD spectra of the thermally stressed rhFVIII samples did not, however, suggest significant differences in protein conformation. To identify conformational changes in rhFVIII that may be involved in rhFVIII aggregation, temperature and solutes were used to perturb the native structure of rhFVIII. Far-UV CD and FTIR studies of rhFVIII as a function of temperature revealed conformational changes corresponding to an increase in intermolecular beta-sheet content beginning at approximately 45 degrees C with significant aggregation observed above 60 degrees C. Fluorescence and DSC studies of rhFVIII also indicated conformational changes initiating between 45 and 50 degrees C. An increase in the exposure of hydrophobic surfaces was observed beginning at approximately 40 degrees C, as monitored by increased binding of the fluorescent probe, bis-anilinonaphthalene sulfonic acid (bis-ANS). Perturbation by various solutes produced several transitions prior to extensive unfolding of rhFVIII. In all cases, a common transition, characterized by an increase in the wavelength of the fluorescence emission maximum of rhFVIII from approximately 330 to 335 nm, was observed during thermal and solute perturbation of factor VIII. Moreover, this transition was correlated with an increased association of factor VIII upon incubation at 37 degrees C in the presence of various solutes. These results suggest that association of rhFVIII in solution was initiated by a small transition in the tertiary structure of the protein which produced a nucleating species that led to the formation of inactive soluble aggregates.
Hsc70 and gp96 are two heat shock proteins with molecular chaperone and immune-related activities... more Hsc70 and gp96 are two heat shock proteins with molecular chaperone and immune-related activities. The dynamic conformational properties of heat shock proteins appear to play a critical role in their biological activities. In this study, we investigated the effects of pH and temperature on the conformational states of Hsc70 and gp96. The quaternary, tertiary, and secondary structures of both proteins are evaluated by a variety of spectroscopic techniques, including far-UV circular dichroism, Trp fluorescence, ANS fluorescence, and derivative UV absorption spectroscopy. The results are summarized and compared employing an empirical phase diagram approach. Very similar behaviors are seen for both proteins despite their differences in sequence and tertiary structure. Both proteins show substantial conformational lability in responses to the pH and temperature changes of their environment. This study suggests a natural selection for related functional properties through common conformat...
Heavy metals have been implicated in the aggregation of proteins and the pathophysiology of sever... more Heavy metals have been implicated in the aggregation of proteins and the pathophysiology of several neurodegenerative diseases. Herein, we describe the interaction of recombinant human factor VIII (rhFVIII) with Al(+3), Tb(+3), Co(+2), and Fe(+3) using a combination of intrinsic fluorescence, circular dichroism, and high-resolution fourth-derivative absorbance analysis. rhFVIII in solution was titrated with the metal cations and the properties of the resulting complexes were examined. rhFVIII has a tendency to aggregate and inactivate slowly over time under physiological conditions, but this aggregation process is greatly accelerated in the presence of metals with Al(+3) being the most efficient. This leads to a complete loss of activity of the protein. Al(+3)-induced conformational changes in the protein were small but detectable with limited changes seen in secondary and tertiary structure. Because rhFVIII is a multidomain protein with subunits linked through divalent metal cations, the small intramolecular changes seen may be attributed to rearrangements of the subunits to an aggregation-competent conformer that is very similar to that of the native form.
Factor VIII (FVIII), a plasma glycoprotein, is an essential cofactor in the blood coagulation cas... more Factor VIII (FVIII), a plasma glycoprotein, is an essential cofactor in the blood coagulation cascade. It is a multidomain protein, known to bind to phosphatidylserine (PS)-containing membranes. Based on X-ray and electron crystallography data, binding of FVIII to PS-containing membranes has been proposed to occur only via the C2 domain. Based on these models, the molecular topology of membrane-bound FVIII
Nature structural & molecular biology, Jan 23, 2015
Immunoglobulin G4 antibodies exhibit unusual properties with important biological consequences. W... more Immunoglobulin G4 antibodies exhibit unusual properties with important biological consequences. We report the structure of the human full-length IgG4 S228P anti-PD1 antibody pembrolizumab, solved to 2.3-Å resolution. Pembrolizumab is a compact molecule, consistent with the presence of a short hinge region. The Fc domain is glycosylated at the CH2 domain on both chains, but one CH2 domain is rotated 120° with respect to the conformation observed in all reported structures to date, and its glycan chain faces the solvent. We speculate that this new conformation is driven by the shorter hinge. The structure suggests a role for the S228P mutation in preventing the IgG4 arm exchange. In addition, this unusual Fc conformation suggests possible structural diversity between IgG subclasses and shows that use of isolated antibody fragments could mask potentially important interactions, owing to molecular flexibility.
The presence of aggregates of Factor VIII (FVIII) was assessed for a highly purified form (rFVIII... more The presence of aggregates of Factor VIII (FVIII) was assessed for a highly purified form (rFVIII) and for the formulated preparation containing human serum albumin (rFVIIIf). Size-exclusion chromatography (SEC) on Sepharose CL-6B and TSK 4000 matrices under native conditions revealed < 1% aggregates of FVIII in either rFVIII or rFVIIIf, as determined by FVIII immunoassay of chromatographic fractions. No degradation products were observed. The immunoassay was capable of detecting FVIII immunoreactivity to levels of approximately 5 ng/ml. Overlap of FVIII clotting (FVIII:c) and immunoassay (FVIII:cAg) activities was observed for SEC fractions. Heat stressing of rFVIIIf (47.5 degrees C for 24 h) resulted in a quantitative increase in FVIII-positive material in the void volume of a TSK 4000 column, demonstrating that aggregates of FVIII can be produced and detected by this method. We conclude that aggregates of FVIII in rFVIII and rFVIIIf constitute < or = 1% by the method descri...
Aggregation of proteins is a major problem in their use as drugs and is also involved in a variet... more Aggregation of proteins is a major problem in their use as drugs and is also involved in a variety of pathological diseases. In this study, biophysical techniques were employed to investigate aggregate formation in the pharmaceutically important protein, recombinant human factor VIII (rhFVIII). Recombinant human factor VIII incubated in solution at 37 degrees C formed soluble aggregates as detected by molecular sieve chromatography and dynamic light scattering. This resulted in a corresponding loss of biological activity. Fluorescence and CD spectra of the thermally stressed rhFVIII samples did not, however, suggest significant differences in protein conformation. To identify conformational changes in rhFVIII that may be involved in rhFVIII aggregation, temperature and solutes were used to perturb the native structure of rhFVIII. Far-UV CD and FTIR studies of rhFVIII as a function of temperature revealed conformational changes corresponding to an increase in intermolecular beta-sheet content beginning at approximately 45 degrees C with significant aggregation observed above 60 degrees C. Fluorescence and DSC studies of rhFVIII also indicated conformational changes initiating between 45 and 50 degrees C. An increase in the exposure of hydrophobic surfaces was observed beginning at approximately 40 degrees C, as monitored by increased binding of the fluorescent probe, bis-anilinonaphthalene sulfonic acid (bis-ANS). Perturbation by various solutes produced several transitions prior to extensive unfolding of rhFVIII. In all cases, a common transition, characterized by an increase in the wavelength of the fluorescence emission maximum of rhFVIII from approximately 330 to 335 nm, was observed during thermal and solute perturbation of factor VIII. Moreover, this transition was correlated with an increased association of factor VIII upon incubation at 37 degrees C in the presence of various solutes. These results suggest that association of rhFVIII in solution was initiated by a small transition in the tertiary structure of the protein which produced a nucleating species that led to the formation of inactive soluble aggregates.
Hsc70 and gp96 are two heat shock proteins with molecular chaperone and immune-related activities... more Hsc70 and gp96 are two heat shock proteins with molecular chaperone and immune-related activities. The dynamic conformational properties of heat shock proteins appear to play a critical role in their biological activities. In this study, we investigated the effects of pH and temperature on the conformational states of Hsc70 and gp96. The quaternary, tertiary, and secondary structures of both proteins are evaluated by a variety of spectroscopic techniques, including far-UV circular dichroism, Trp fluorescence, ANS fluorescence, and derivative UV absorption spectroscopy. The results are summarized and compared employing an empirical phase diagram approach. Very similar behaviors are seen for both proteins despite their differences in sequence and tertiary structure. Both proteins show substantial conformational lability in responses to the pH and temperature changes of their environment. This study suggests a natural selection for related functional properties through common conformat...
Heavy metals have been implicated in the aggregation of proteins and the pathophysiology of sever... more Heavy metals have been implicated in the aggregation of proteins and the pathophysiology of several neurodegenerative diseases. Herein, we describe the interaction of recombinant human factor VIII (rhFVIII) with Al(+3), Tb(+3), Co(+2), and Fe(+3) using a combination of intrinsic fluorescence, circular dichroism, and high-resolution fourth-derivative absorbance analysis. rhFVIII in solution was titrated with the metal cations and the properties of the resulting complexes were examined. rhFVIII has a tendency to aggregate and inactivate slowly over time under physiological conditions, but this aggregation process is greatly accelerated in the presence of metals with Al(+3) being the most efficient. This leads to a complete loss of activity of the protein. Al(+3)-induced conformational changes in the protein were small but detectable with limited changes seen in secondary and tertiary structure. Because rhFVIII is a multidomain protein with subunits linked through divalent metal cations, the small intramolecular changes seen may be attributed to rearrangements of the subunits to an aggregation-competent conformer that is very similar to that of the native form.
Factor VIII (FVIII), a plasma glycoprotein, is an essential cofactor in the blood coagulation cas... more Factor VIII (FVIII), a plasma glycoprotein, is an essential cofactor in the blood coagulation cascade. It is a multidomain protein, known to bind to phosphatidylserine (PS)-containing membranes. Based on X-ray and electron crystallography data, binding of FVIII to PS-containing membranes has been proposed to occur only via the C2 domain. Based on these models, the molecular topology of membrane-bound FVIII
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Papers by Ramesh Kashi