Samuel et al. 10.1073/pnas.0703080104. |
Fig. 6. Alternative solution structure of FXI A4. (a) The backbone overlay of 14 low-energy structures of FXI A4 in an alternative conformer of the mobile loop (residues 316-325). Colored segments indicate b-sheet (blue) and helices (red), respectively. (b) Ribbon representation of the two domains of the dimer using the coloring scheme of Fig. 1b.
Fig. 7. Sequence alignment of apple domains and A4 dimer interface. (a) Amino acid sequence alignment of FXI apple domains. The hydrophobic doublets involved in dimer formation (red boxes) are unique to the A4 domain. The blue box shows residues in the flexible loop that adopts multiple conformations. Proline residues at the beginning of this sequence and typical turn sequence -GXGX- (bold) are only observed in A4 domain. In other domains, the proline residues are involved in b-turns close to the apex of the loop. (b) Dimer interface of FXI A4. Only one of the two monomers is shown with the dimer interface facing up. In this view, the twofold symmetry axis of the dimer lies vertically across the center of the molecule so that residues in the left half of the interface interact with residues in the right half of the other monomer.
Fig. 8. NMR evidence for conformational heterogeneity in A4 involving a mobile loop (residues 316-325). Temperature-dependent changes in peak height for selected 1H-15N cross-peaks in the HSQC spectrum of A4 recorded over a range of temperatures (10-45°C). Most residues exhibit a monotonous increase in cross peak intensity with increasing temperature (e.g., K340) reflecting the temperature dependence of solvent viscosity. In contrast, some of the cross peaks assigned to residues in the mobile loop show anomalous temperature dependence, such as increasing intensity with decreasing temperature (e.g., N322), or a bell-shaped behavior (S320 and E323). Other residues (e.g., C321) have very low intensity throughout the observed temperature range. This unusual temperature dependence indicates that the dynamics of exchange between alternative loop conformations approaches the intermediate-exchange regime at higher temperatures, resulting in severe line broadening. At lower temperature, some of the peaks gain intensity as the exchange rate slows relative to the chemical shift difference between the two conformational states.
Fig. 9. Deglycosylation of factor XI. SDS/PAGE analysis of untreated (glycosylated) samples of FXI and FXIa (lanes 1 and 3), and deglycosylated samples of FXI and FXIa (lanes 2 and 4) obtained by overnight treatment with the endo glycosidase PNGase F under native conditions. Identical gel shifts were observed for samples that were heat-denatured in the presence of SDS before incubation with PNGase F (data not shown), indicating that complete deglycosylation is achieved under native conditions. Functional characterization of deglycosylated FXIa: The specific activity of FXI before treatment with N-glycosidase was 242 units/mg, whereas after deglycosylation, the specific activity was 213 units/mg. This 12% difference is considered to be within the range of variation of both the functional assay of FXI activity and the protein assay. We conclude that the absence of carbohydrate from FXI does not have an apparent effect on its functional activity.
Fig. 10. Modeling the domain rearrangements associated with FXI zymogen activation on the basis of SAXS experiments. (a) Logarithmic plot of the measured scattering intensity, I(Q), vs. scattering vector, Q, for FXIa. The heavy line represents the predicted scattering profiles calculated using the program CRISOL (1), based on the model of FXa depicted in the b. (b) Model of FXIa obtained by iterative fitting of the SAXS data in a, using the NMR structure of the A4 dimer as a starting point. The FXIa dimer is shown in surface-rendered representation colored according to domains, as indicated. (c) Crystal structure of the FXI zymogen (2). The domains are shown using the same coloring scheme as in b.
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SI Materials and Methods
NMR Spectroscopy.
NMR data were collected on a Bruker DMX-600 spectrometer equipped with a 5-mm x, y, z-shielded pulsed-field gradient triple-resonance probe (Bruker Biospin GmbH, Rheinstetten, Germany). Felix (Felix NMR, Inc., San Diego, CA) and Sparky (www.cgl.ucsf.edu/home/sparky) were used for processing and resonance assignment, respectively. Chemical shift values for 1H, 13C and 15N were assigned on the basis of CBCANH CBCA(CO)NH, 15N-HSQC, 15N-HSQC-TOCSY, and HNHA experiments (1-3). Side-chain assignments were obtained from 3D-15N TOCSY-HSQC (4) and 3D-HCCH-TOCSY (5). Distance restraints were obtained from cross-peak volumes in 15N-edited and 13C-edited NOESY-HSQC (6, 7) and homonuclear NOESY (8). Intersubunit NOEs were assigned using an iterative procedure. A chain-selective labeling strategy to identify intermonomer NOEs (9) was unsuccessful in the case of the covalently linked A4 dimer, since the presence of an intermolecular disulfide bond makes it difficult to prepare mixed (15N-labeled and unlabeled) dimers. Hydrogen bonds were assigned to 30 slowly exchanging amide protons from a total of 50 peaks observed in an HSQC spectrum recorded on a fresh solution of A4 in 2H2O (pD 6, 37°C). Axially compressed acrylamide gel (7%) was prepared following a published protocol (10). The internuclear dipolar couplings (in Hz) were determined from the difference in J-splitting between the isotropic and aligned phases (≈5-10% gel compression). Initial values for the axial (Aa) and rhombic (Ar) components of the alignment tensor A were obtained from a histogram of the dipolar coupling that were normalized as described in ref. 11. Structure calculations were performed on a Linux Red Hat 8.0 workstation, using a hybrid distance geometry simulated annealing protocol implemented in CNS1.1 (http://cns.csb.yale.edu). Initial structures were calculated using NOE-based distance constraints, dihedral angle and hydrogen bond restraints, and further refined on the basis of residual dipolar coupling restraints, using standard annealing protocols. From a total of 100 structures generated, 35 were selected with rmsd for bond lengths <0.01 Å, for bond angles <1.0°, and for improper bond angles <1.0°. The 14 low-energy structures displayed in Fig. 1a have no NOE violations >0.5 Å and no dihedral angle violations >5°. Graphical displays of structures were generated using Chimera (www.cgl.ucsf.edu/chimera).Solution Scattering Experiments.
SAXS experiments were performed at room temperature with an in-house apparatus consisting of a Rigaku 007 microfocus rotating anode x-ray generator (Rigaku/MSC) and a multiwire detector (Molecular Metrology, Inc), as described by Bu et al. (12). The optical configuration used allows a Q range from 0.013 to 0.30 Å-1 to be measured, where Q = (4p sin(q / 2) / l) is the magnitude of the scattering vector, q is the scattering angle, and l = 1.54 Å is the wavelength of the x-ray. The scattering patterns were circularly averaged, and the reduced scattering data were plotted as scattering intensity I(Q) vs. Q profiles. The radius of gyration, Rg, was obtained from the Guinier approximation by linear least squares fitting in the QRg < 1 region, where the forward scattering intensity I(0) is proportional to the molecular weight of the protein complex (13). Inverse Fourier transformation of I(Q) gives the length distribution function P(r). P(r) functions were generated by the program GNOM (14), which also yields Rg values. Low-resolution molecular envelopes were reconstructed from the experimental SAXS data using the program DAMMIN (15), which employs simulated annealing to determine envelope functions that are parameterized in terms of spherical harmonics. The constraints used included twofold symmetry of the FXI/FXIa dimer in addition to positive scattering density and compactness. The PDB-formatted dummy bead coordinates generated by DAMMIN were converted into a 3D molecular envelope using the program SITUS (16). Additional details can be found in Ho et al. (17). DLS measurements were performed on a Wyatt technologies DynaPro Titan molecular sizing system at 23°C operating at a wavelength of 824.7 nm. The data were analyzed by using DYNAMICS V6.Molecular Modeling.
The NMR structure of the A4 domain (PDB ID code 2J8L) and the crystal structure of the complex of the FXIa catalytic domain with KPI (PDB ID code 1ZJD) were used for molecular modeling of possible domain rearrangements associated with zymogen activation, using Chimera (18). The serine mutation at position 482 in the catalytic domain was reverted to the wild-type residue cysteine. In the A4 structure a cysteine residue was added to the C terminus at position 362 to allow formation of an interchain disulfide bond with Cys-482. For the model in Fig. 5 the catalytic domains were rotated around the disulfide bond to explore various sterically allowed conformations.1. Montelione GT, Wagner G (1990) J Magn Reson 87:183-188.
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