Dataset Information

Dimer dissociation and unfolding mechanism of coagulation factor XI apple 4 domain: spectroscopic and mutational analysis.

ABSTRACT: The blood coagulation protein factor XI (FXI) consists of a pair of disulfide-linked chains each containing four apple domains and a catalytic domain. The apple 4 domain (A4; F272-E362) mediates non-covalent homodimer formation even when the cysteine involved in an intersubunit disulfide is mutated to serine (C321S). To understand the role of non-covalent interactions stabilizing the FXI dimer, equilibrium unfolding of wild-type A4 and its C321S variant was monitored by circular dichroism, intrinsic tyrosine fluorescence and dynamic light scattering measurements as a function of guanidine hydrochloride concentration. Global analysis of the unimolecular unfolding transition of wild-type A4 revealed a partially unfolded equilibrium intermediate at low to moderate denaturant concentrations. The optically detected equilibrium of C321S A4 also fits best to a three-state model in which the native dimer unfolds via a monomeric intermediate state. Dimer dissociation is characterized by a dissociation constant, K(d), of approximately 90 nM (in terms of monomer), which is in agreement with the dissociation constant measured independently using fluorescence anisotropy. The results imply that FXI folding occurs via a monomeric equilibrium intermediate. This observation sheds light on the effect of certain naturally occurring mutations, such as F283L, which lead to intracellular accumulation of non-native forms of FXI. To investigate the structural and energetic consequences of the F283L mutation, which perturbs a cluster of aromatic side-chains within the core of the A4 monomer, it was introduced into the dissociable dimer, C321S A4. NMR chemical shift analysis confirmed that the mutant can assume a native-like dimeric structure. However, equilibrium unfolding measurements show that the mutation causes a fourfold increase in the K(d) value for dissociation of the native dimer and a 1 kcal/mol stabilization of the monomer, resulting in a highly populated intermediate. Since the F283 side-chain does not directly participate in the dimer interface, we propose that the F283L mutation leads to increased dimer dissociation by stabilizing a monomeric state with altered side-chain packing that is unfavorable for homodimer formation.

SUBMITTER: Riley PW

PROVIDER: S-EPMC1945241 | biostudies-literature | 2007 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Dimer dissociation and unfolding mechanism of coagulation factor XI apple 4 domain: spectroscopic and mutational analysis.

Riley Paul W PW Cheng Hong H Samuel Dharmaraj D Roder Heinrich H Walsh Peter N PN

Journal of molecular biology 20061229 2

The blood coagulation protein factor XI (FXI) consists of a pair of disulfide-linked chains each containing four apple domains and a catalytic domain. The apple 4 domain (A4; F272-E362) mediates non-covalent homodimer formation even when the cysteine involved in an intersubunit disulfide is mutated to serine (C321S). To understand the role of non-covalent interactions stabilizing the FXI dimer, equilibrium unfolding of wild-type A4 and its C321S variant was monitored by circular dichroism, intri ...[more]

PMID: 17257616

Similar Datasets

Project description:Essentials Zymogen PK is activated to PKa and cleaves substrates kininogen and FXII contributing to bradykinin generation. Monomeric PKa and dimeric homologue FXI utilize the N-terminal apple domains to recruit substrates. A high-resolution 1.3 Å structure of full-length PKa reveals an active conformation of the protease and apple domains. The PKa protease and four-apple domain disc organization is 180° rotated compared to FXI. SUMMARY: Background Plasma prekallikrein (PK) and factor XI (FXI) are apple domain-containing serine proteases that when activated to PKa and FXIa cleave substrates kininogen, factor XII, and factor IX, respectively, directing plasma coagulation, bradykinin release, inflammation, and thrombosis pathways. Objective To investigate the three-dimensional structure of full-length PKa and perform a comparison with FXI. Methods A series of recombinant full-length PKa and FXI constructs and variants were developed and the crystal structures determined. Results and conclusions A 1.3 Å structure of full-length PKa reveals the protease domain positioned above a disc-shaped assemblage of four apple domains in an active conformation. A comparison with the homologous FXI structure reveals the intramolecular disulfide and structural differences in the apple 4 domain that prevents dimer formation in PK as opposed to FXI. Two latchlike loops (LL1 and LL2) extend from the PKa protease domain to form interactions with the apple 1 and apple 3 domains, respectively. A major unexpected difference in the PKa structure compared to FXI is the 180° disc rotation of the apple domains relative to the protease domain. This results in a switched configuration of the latch loops such that LL2 interacts and buries portions of the apple 3 domain in the FXI zymogen whereas in PKa LL2 interacts with the apple 1 domain. Hydrogen-deuterium exchange mass spectrometry on plasma purified human PK and PKa determined that regions of the apple 3 domain have increased surface exposure in PKa compared to the zymogen PK, suggesting conformational change upon activation.

Project description:Among Mononegavirales, the Pneumovirus family stands out by its RNA polymerase processivity that relies on a transcription antiterminator, the M2-1 protein, which also plays a key role in viral particle assembly. Biophysical and structural evidence shows that this RNA-binding tetramer is strongly modulated by a CCCH Zn2+ binding motif. We show that while the global dissociation/unfolding free energy is 10 kcal mol-1, more stable for the respiratory syncytial virus M2-1, the human metapneumovirus (HMPV) counterpart shows a 7 kcal mol-1 higher intersubunit affinity. Removal of Zn2+ from both homologues leads to an apo-monomer of identical secondary structure that further undergoes a slow irreversible oligomerization. Mutation of the histidine residue of the Zn2+ motif to cysteine or alanine leads directly to large oligomers, strongly suggesting that metal coordination has an exquisite precision for modulating the quaternary arrangement. Zn2+ removal is very slow and requires subdenaturing concentrations of guanidine chloride, suggesting a likely local folding energy barrier. Exploring a broad combination of denaturant and ethylenediaminetetraacetic acid conditions, we showed that the metapneumovirus protein has to overcome a higher energy barrier to trigger Zn2+ removal-driven dissociation, in concordance with a slower dissociation kinetics. In silico modeling of open and close conformations for both M2-1 tetramers together with interaction energy calculations reveals that the gradual opening of protomers decreases the number of intersubunit contacts. Half of the interaction energy holding each protomer in the tetramer comes from the CCCH motif, while HMPV-M2-1 harbors additional contacts between the CCCH motif of one subunit and the core domain of a protomer located in trans, allowing the rationalization of the experimental data obtained. Overall, the evidence points at a key role of the CCCH motif in switching between structural and consequently functional alternatives of the M2-1 protein.

Project description:High levels of factor XI (FXI) increase the risk of thromboembolic disease. However, the genetic and environmental factors regulating FXI expression are still largely unknown. The aim of our study was to evaluate the regulation of FXI by microRNAs (miRNAs) in the human liver. In silico prediction yielded four miRNA candidates that might regulate FXI expression. HepG2 cells were transfected with miR-181a-5p, miR- 23a-3p, miR-16-5p and miR-195-5p. We used mir-494, which was not predicted to bind to F11, as a negative control. Only miR-181a-5p caused a significant decrease both in FXI protein and F11 mRNA levels. In addition, transfection with a miR-181a-5p inhibitor in PLC/PRF/5 hepatic cells increased both the levels of F11 mRNA and extracellular FXI. Luciferase assays in human colon cancer cells deficient for Dicer (HCT-DK) demonstrated a direct interaction between miR-181a-5p and 3'untranslated region of F11. Additionally, F11 mRNA levels were inversely and significantly correlated with miR-181a-5p levels in 114 healthy livers, but not with miR-494. This study demonstrates that FXI expression is directly regulated by a specific miRNA, miR-181a-5p, in the human liver. Future studies are necessary to further investigate the potential consequences of miRNA dysregulation in pathologies involving FXI. In the study presented here, we report arrays including 1,898 human mature miRNAs (LCSciences, Houston, TX; Sanger mirBase Release 18.0) using total RNA extracted from a healthy human liver sample E122; a Caucasian donor that gave informed consent were provided by biobanc CIBERehd. Human liver studies were apporved by Local Ethics Committee from Hospital Universitario Morales Meseguer in Murcia. In the study presented here, we report arrays including 2,019 human mature miRNAs (LCSciences, Houston, TX; Sanger mirBase Release 19.0) using total RNA extracted from 3 healthy human liver samples (662, 664, 771) from Caucasian donors that gave informed consent were provided by St. Jude Liver Resource at St. Distribution System and by cooperative Human Tissue Network. Human liver studies were apporved by Local Ethics Committee from Hospital Universitario Morales Meseguer in Murcia

Dataset Information

Dimer dissociation and unfolding mechanism of coagulation factor XI apple 4 domain: spectroscopic and mutational analysis.

Publications

Dimer dissociation and unfolding mechanism of coagulation factor XI apple 4 domain: spectroscopic and mutational analysis.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets