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1.
Anal Chem ; 87(12): 5973-80, 2015 Jun 16.
Article in English | MEDLINE | ID: mdl-25978680

ABSTRACT

Human growth hormone (hGH), and its receptor interaction, is essential for cell growth. To stabilize a flexible loop between helices 3 and 4, while retaining affinity for the hGH receptor, we have engineered a new hGH variant (Q84C/Y143C). Here, we employ hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map the impact of the new disulfide bond on the conformational dynamics of this new hGH variant. Compared to wild type hGH, the variant exhibits reduced loop dynamics, indicating a stabilizing effect of the introduced disulfide bond. Furthermore, the disulfide bond exhibits longer ranging effects, stabilizing a short α-helix quite distant from the mutation sites, but also rendering a part of the α-helical hGH core slightly more dynamic. In the regions where the hGH variant exhibits a different deuterium uptake than the wild type protein, electron transfer dissociation (ETD) fragmentation has been used to pinpoint the residues responsible for the observed differences (HDX-ETD). Finally, by use of surface plasmon resonance (SPR) measurements, we show that the new disulfide bond does not compromise receptor affinity. Our work highlight the analytical potential of HDX-ETD combined with functional assays to guide protein engineering.


Subject(s)
Disulfides/chemistry , Human Growth Hormone/chemistry , Protein Engineering , Deuterium Exchange Measurement , Electron Transport , Humans , Mass Spectrometry , Models, Molecular , Protein Conformation
2.
Blood ; 118(8): 2333-41, 2011 Aug 25.
Article in English | MEDLINE | ID: mdl-21700771

ABSTRACT

Current management of hemophilia B entails multiple weekly infusions of factor IX (FIX) to prevent bleeding episodes. In an attempt to make a longer acting recombinant FIX (rFIX), we have explored a new releasable protraction concept using the native N-glycans in the activation peptide as sites for attachment of polyethylene glycol (PEG). Release of the activation peptide by physiologic activators converted glycoPEGylated rFIX (N9-GP) to native rFIXa and proceeded with normal kinetics for FXIa, while the K(m) for activation by FVIIa-tissue factor (TF) was increased by 2-fold. Consistent with minimal perturbation of rFIX by the attached PEG, N9-GP retained 73%-100% specific activity in plasma and whole-blood-based assays and showed efficacy comparable with rFIX in stopping acute bleeds in hemophilia B mice. In animal models N9-GP exhibited up to 2-fold increased in vivo recovery and a markedly prolonged half-life in mini-pig (76 hours) and hemophilia B dog (113 hours) compared with rFIX (16 hours). The extended circulation time of N9-GP was reflected in prolonged correction of coagulation parameters in hemophilia B dog and duration of effect in hemophilia B mice. Collectively, these results suggest that N9-GP has the potential to offer efficacious prophylactic and acute treatment of hemophilia B patients at a reduced dosing frequency.


Subject(s)
Factor IX/chemistry , Factor IX/metabolism , Animals , Binding Sites , Disease Models, Animal , Dogs , Factor IX/genetics , Female , Half-Life , Hemophilia B/blood , Hemophilia B/drug therapy , Hemophilia B/genetics , Hemostatics/blood , Hemostatics/chemistry , Hemostatics/pharmacology , Humans , In Vitro Techniques , Kinetics , Male , Mice , Mice, Mutant Strains , Polyethylene Glycols/chemistry , Polysaccharides/chemistry , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , Swine , Swine, Miniature
3.
J Biol Chem ; 284(38): 26137-48, 2009 Sep 18.
Article in English | MEDLINE | ID: mdl-19592498

ABSTRACT

Interleukin-1beta (IL-1beta) is a master cytokine involved in initiating the innate immune response in vertebrates (Dinarello, C. A. (1994) FASEB J. 8, 1314-1325). It is first synthesized as an inactive 269-residue precursor (pro-interleukin-1beta or pro-IL-1beta). Pro-IL-1beta requires processing by caspase-1 to generate the active, mature 153-residue cytokine. In this study, we combined hydrogen/deuterium exchange mass spectrometry, circular dichroism spectroscopy, and enzymatic digestion comparative studies to investigate the configurational landscape of pro-IL-1beta and the role the N terminus plays in modulating the landscape. We find that the N terminus keeps pro-IL-1beta in a protease-labile state while maintaining a core region of stability in the C-terminal region, the eventual mature protein. In mature IL-1beta, this highly protected region maps back to the area protected earliest in the NMR studies characterizing an on-route kinetic refolding intermediate. This protected region also encompasses two important functional loops that participate in the IL-1beta/receptor binding interface required for biological activity. We propose that the purpose of the N-terminal precursor region in pro-IL-1beta is to suppress the function of the eventual mature region while keeping a structurally and also functionally important core region primed for the final folding into the native, active state of the mature protein. The presence of the self-inhibiting precursor region provides yet another layer of regulation in the life cycle of this important cytokine.


Subject(s)
Interleukin-1/chemistry , Interleukin-1beta/chemistry , Protein Precursors/chemistry , Deuterium Exchange Measurement/methods , Humans , Interleukin-1/genetics , Interleukin-1/metabolism , Interleukin-1beta/genetics , Interleukin-1beta/metabolism , Protein Precursors/genetics , Protein Precursors/metabolism , Protein Structure, Secondary/physiology , Protein Structure, Tertiary/physiology , Structure-Activity Relationship
4.
J Biol Chem ; 283(27): 19085-94, 2008 Jul 04.
Article in English | MEDLINE | ID: mdl-18467331

ABSTRACT

The crystal structure of the complex between an N-terminally truncated G129R human prolactin (PRL) variant and the extracellular domain of the human prolactin receptor (PRLR) was determined at 2.5A resolution by x-ray crystallography. This structure represents the first experimental structure reported for a PRL variant bound to its cognate receptor. The binding of PRL variants to the PRLR extracellular domain was furthermore characterized by the solution state techniques, hydrogen exchange mass spectrometry, and NMR spectroscopy. Compared with the binding interface derived from mutagenesis studies, the structural data imply that the definition of PRL binding site 1 should be extended to include residues situated in the N-terminal part of loop 1 and in the C terminus. Comparison of the structure of the receptor-bound PRL variant with the structure reported for the unbound form of a similar analogue ( Jomain, J. B., Tallet, E., Broutin, I., Hoos, S., van Agthoven, J., Ducruix, A., Kelly, P. A., Kragelund, B. B., England, P., and Goffin, V. (2007) J. Biol. Chem. 282, 33118-33131 ) demonstrates that receptor-induced changes in the backbone of the four-helix bundle are subtle, whereas large scale rearrangements and structuring occur in the flexible N-terminal part of loop 1. Hydrogen exchange mass spectrometry data imply that the dynamics of the four-helix bundle in solution generally become stabilized upon receptor interaction at binding site 1.


Subject(s)
Peptides/chemistry , Prolactin/chemistry , Receptors, Prolactin/antagonists & inhibitors , Receptors, Prolactin/chemistry , Amino Acid Substitution , Binding Sites/genetics , Crystallography, X-Ray , Humans , Mutagenesis , Nuclear Magnetic Resonance, Biomolecular , Peptides/genetics , Peptides/metabolism , Prolactin/genetics , Prolactin/metabolism , Protein Structure, Quaternary , Protein Structure, Secondary , Protein Structure, Tertiary/genetics , Receptors, Prolactin/genetics , Receptors, Prolactin/metabolism
5.
J Biol Chem ; 283(19): 13378-87, 2008 May 09.
Article in English | MEDLINE | ID: mdl-18343822

ABSTRACT

Factor VIIa (FVIIa) circulates in the blood in a zymogen-like state. Only upon association with membrane-bound tissue factor (TF) at the site of vascular injury does FVIIa become active and able to initiate blood coagulation. Here we used hydrogen exchange monitored by mass spectrometry to investigate the conformational effects of site-directed mutagenesis at key positions in FVIIa and the origins of enhanced intrinsic activity of FVIIa analogs. The differences in hydrogen exchange of two highly active variants, FVIIa(DVQ) and FVIIa(VEAY), imply that enhanced catalytic efficiency was attained by two different mechanisms. Regions protected from exchange in FVIIa(DVQ) include the N-terminal tail and the activation pocket, which is a subset of the regions of FVIIa protected from exchange upon TF binding. FVIIa(DVQ) appeared to adopt an intermediate conformation between the free (zymogen-like) and TF-bound (active) form of FVIIa and to attain enhanced activity by partial mimicry of TF-induced activation. In contrast, exchange-protected regions in FVIIa(VEAY) were confined to the vicinity of the active site of FVIIa. Thus, the changes in FVIIa(VEAY) appeared to optimize the active site region rather than imitate the TF-induced effect. Hydrogen exchange analysis of the FVIIa(M306D) variant, which was unresponsive to stimulation by TF, correlated widespread reductions in exchange to the single mutation in the TF-binding region. These results reveal the delicate interplay between key allosteric sites necessary to achieve the transition of FVIIa into the active form.


Subject(s)
Allosteric Site , Factor VIIa/chemistry , Factor VIIa/metabolism , Hydrogen/chemistry , Amino Acid Sequence , Binding Sites , Crystallography, X-Ray , Factor VIIa/genetics , Humans , Mass Spectrometry , Models, Molecular , Molecular Sequence Data , Mutation/genetics , Protein Binding , Protein Structure, Quaternary , Protein Structure, Tertiary , Thromboplastin/chemistry , Thromboplastin/metabolism
6.
J Biol Chem ; 281(32): 23018-24, 2006 Aug 11.
Article in English | MEDLINE | ID: mdl-16687401

ABSTRACT

Coagulation factor VIIa (FVIIa) is a serine protease that, after binding to tissue factor (TF), plays a pivotal role in the initiation of blood coagulation. We used hydrogen exchange monitored by mass spectrometry to visualize the details of FVIIa activation by comparing the exchange kinetics of distinct molecular states, namely zymogen FVII, endoproteolytically cleaved FVIIa, TF-bound zymogen FVII, TF-bound FVIIa, and FVIIa in complex with an active site inhibitor. The hydrogen exchange kinetics of zymogen FVII and FVIIa are identical indicating highly similar solution structures. However, upon tissue factor binding, FVIIa undergoes dramatic structural stabilization as indicated by decreased exchange rates localized throughout the protease domain and in distant parts of the light chain, spanning across 50A and revealing a concerted interplay between functional sites in FVIIa. The results provide novel insights into the cofactor-induced activation of this important protease and reveal the potential for allosteric regulation in the trypsin family of proteases.


Subject(s)
Factor VIIa/chemistry , Factor VIIa/metabolism , Hydrogen/chemistry , Allosteric Site , Enzyme Activation , Enzyme Precursors/chemistry , Humans , Kinetics , Mass Spectrometry/methods , Models, Molecular , Molecular Conformation , Peptide Hydrolases/chemistry , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Trypsin/chemistry
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