Large Conformational Differences Research Articles

Near-atomic structure of an atadenovirus reveals a conserved capsid-binding motif and intergenera variations in cementing proteins.

Of five known adenovirus genera, high-resolution structures are available only for mammalian-infecting mastadenoviruses. We present the first high-resolution structure of an adenovirus with nonmammalian host: lizard atadenovirus LAdV-2. We find a large conformational difference in the internal vertex protein IIIa between mast- and atadenoviruses, induced by the presence of an extended polypeptide. This polypeptide, and α-helical clusters beneath the facet, likely correspond to genus-specific proteins LH2 and p32k. Another genus-specific protein, LH3, with a fold typical of bacteriophage tailspikes, contacts the capsid surface via a triskelion structure identical to that used by mastadenovirus protein IX, revealing a conserved capsid-binding motif and an ancient gene duplication event. Our data also suggest that mastadenovirus E1B-55 K was exapted from the atadenovirus-like LH3 protein. This work provides new information on the evolution of adenoviruses, emphasizing the importance of minor coat proteins for determining specific physicochemical properties of virions and most likely their tropism.

Architecture and structural dynamics of the heteromeric GluK2/K5 kainate receptor.

Kainate receptors (KARs) are L-glutamate-gated ion channels that regulate synaptic transmission and modulate neuronal circuits. KARs have strict assembly rules and primarily function as heteromeric receptors in the brain. A longstanding question is how KAR heteromer subunits organize and coordinate together to fulfill their signature physiological roles. Here we report structures of the GluK2/GluK5 heteromer in apo, antagonist-bound, and desensitized states. The receptor assembles with two copies of each subunit, ligand binding domains arranged as two heterodimers and GluK5 subunits proximal to the channel. Strikingly, during desensitization, GluK2, but not GluK5, subunits undergo major structural rearrangements to facilitate channel closure. We show how the large conformational differences between antagonist-bound and desensitized states are mediated by the linkers connecting the pore helices to the ligand binding domains. This work presents the first KAR heteromer structure, reveals how its subunits are organized, and resolves how the heteromer can accommodate functionally distinct closed channel structures.

Molecular mechanism of point mutation-induced Monopolar spindle 1 (Mps1/TTK) inhibitor resistance revealed by a comprehensive molecular modeling study.

BackgroundMonopolar spindle 1 (Mps1/TTK) is an apical dual-specificity protein kinase in the spindle assembly checkpoint (SAC) that guarantees accurate segregation of chromosomes during mitosis. High levels of Mps1 are found in various types of human malignancies, such as glioblastoma, osteosarcoma, hepatocellular carcinoma, and breast cancer. Several potent inhibitors of Mps1 exist, and exhibit promising activity in many cell cultures and xenograft models. However, resistance due to point mutations in the kinase domain of Mps1 limits the therapeutic effects of these inhibitors. Understanding the detailed resistance mechanism induced by Mps1 point mutations is therefore vital for the development of novel inhibitors against malignancies.MethodsIn this study, conventional molecular dynamics (MD) simulation and Gaussian accelerated MD (GaMD) simulation were performed to elucidate the resistance mechanisms of Cpd-5, a potent Mps1 inhibitor, induced by the four representative mutations I531M, I598F, C604Y, S611R.ResultsOur results from conventional MD simulation combined with structural analysis and free energy calculation indicated that the four mutations weaken the binding affinity of Cpd-5 and the major variations in structural were the conformational changes of the P-loop, A-loop and αC-helix. Energetic differences of per-residue between the WT system and the mutant systems indicated the mutations may allosterically regulate the conformational ensemble and the major variations were residues of Ile-663 and Gln-683, which located in the key loops of catalytic loop and A-loop, respectively. The large conformational and energetic differences were further supported by the GaMD simulations. Overall, these obtained molecular mechanisms will aid rational design of novel Mps1 inhibitors to combat inhibitor resistance.

Packaged and Free Satellite Tobacco Mosaic Virus (STMV) RNA Genomes Adopt Distinct Conformational States.

The RNA genomes of viruses likely undergo multiple functionally important conformational changes during their replication cycles, changes that are poorly understood at present. We used two complementary in-solution RNA structure probing strategies (SHAPE-MaP and RING-MaP) to examine the structure of the RNA genome of satellite tobacco mosaic virus inside authentic virions and in a capsid-free state. Both RNA states feature similar three-domain architectures in which each major replicative function-translation, capsid coding, and genome synthesis-fall into distinct domains. There are, however, large conformational differences between the in-virion and capsid-free states, primarily in one arm of the central T domain. These data support a model in which the packaged capsid-bound RNA is constrained in a local high-energy conformation by the native capsid shell. The removal of the viral capsid then allows the RNA genome to relax into a more thermodynamically stable conformation. The RNA architecture of the central T domain thus likely changes during capsid assembly and disassembly and may play a role in genome packaging.

Calmodulin in complex with the first IQ motif of myosin-5a functions as an intact calcium sensor

The motor function of vertebrate myosin-5a is inhibited by its tail in a Ca2+-dependent manner. We previously demonstrated that the calmodulin (CaM) bound to the first isoleucine-glutamine (IQ) motif (IQ1) of myosin-5a is responsible for the Ca2+-dependent regulation of myosin-5a. We have solved the crystal structure of a truncated myosin-5a containing the motor domain and IQ1 (MD-IQ1) complexed with Ca2+-bound CaM (Ca2+-CaM) at 2.5-Å resolution. Compared with the structure of the MD-IQ1 complexed with essential light chain (an equivalent of apo-CaM), MD-IQ1/Ca2+-CaM displays large conformational differences in IQ1/CaM and little difference in the motor domain. In the MD-IQ1/Ca2+-CaM structure, the N-lobe and the C-lobe of Ca2+-CaM adopt an open conformation and grip the C-terminal and the N-terminal portions of the IQ1, respectively. Remarkably, the interlobe linker of CaM in IQ1/Ca2+-CaM is in a position opposite that in IQ1/apo-CaM, suggesting that CaM flip-flops relative to the IQ1 during the Ca2+ transition. We demonstrated that CaM continuously associates with the IQ1 during the Ca2+ transition and that the binding of CaM to IQ1 increases Ca2+ affinity and substantially changes the kinetics of the Ca2+ transition, suggesting that the IQ1/CaM complex functions as an intact Ca2+ sensor responding to distinct calcium signals.

In Silico Reduction of Conformational Variance in cryo-EM Imaging

One of the biggest challenges in the analysis of cryo-EM images is the heterogeneity and flexibility of the molecules, which severely limits the achievable resolution. We will present our recent work on the development of a computational approach to reduce the conformational variance of a set of single-particle images with the goal of increasing the resolution. We first reconstruct the conformational variance of a molecule from the variance of the density. The information on conformational variance is then used to extensively classify the images into a number of different classes. In a next step, the individual 3D density reconstructions from all classes are recombined into one single (higher resolution) reconstruction by a novel flexible averaging procedure. In the flexible averaging the density grids of two maps are elastically deformed to account for large scale conformational differences thereby reducing the conformational variance of a data set. The goal is to improve the resolution and at the same time to gain a complete picture of the conformational variance of a macromolecule.

The Crystal Structure of the Plant Small GTPase OsRac1 Reveals Its Mode of Binding to NADPH Oxidase

Rac/Rop proteins are Rho-type small GTPases that act as molecular switches in plants. Recent studies have identified these proteins as key components in many major plant signaling pathways, such as innate immunity, pollen tube growth, and root hair formation. In rice, the Rac/Rop protein OsRac1 plays an important role in regulating the production of reactive oxygen species (ROS) by the NADPH oxidase OsRbohB during innate immunity. However, the molecular mechanism by which OsRac1 regulates OsRbohB remains unknown. Here, we report the crystal structure of OsRac1 complexed with the non-hydrolyzable GTP analog guanosine 5'-(β,γ-imido)triphosphate at 1.9 Å resolution; this represents the first active-form structure of a plant small GTPase. To elucidate the ROS production in rice cells, structural information was used to design OsRac1 mutants that displayed reduced binding to OsRbohB. Only mutations in the OsRac1 Switch I region showed attenuated interactions with OsRbohB in vitro. In particular, Tyr(39) and Asp(45) substitutions suppressed ROS production in rice cells, indicating that these residues are critical for interaction with and activation of OsRbohB. Structural comparison of active-form OsRac1 with AtRop9 in its GDP-bound inactive form showed a large conformational difference in the vicinity of these residues. Our results provide new insights into the molecular mechanism of the immune response through OsRac1 and the various cellular responses associated with plant Rac/Rop proteins.

Crystal Growth, Structure, and Polymorphic Behavior of an Ionic Liquid: Phthalate Derivative of N-Butyl,N-methylimidazolium Hexafluorophosphate

After the multistep synthesis of an original imidazolium hexafluorophosphate ionic liquid, [pbmim][PF6], two polymorphic forms were isolated from methanolic solution and characterized by XRPD, DSC, and Raman spectroscopy. Stable Form A (mp 90.3 °C) was obtained by conventional crystallization at a moderate cooling rate (<10 K/min), whereas metastable Form B (mp 83.2 °C) was spontaneously produced from a liquid–liquid demixing (oiling out) when a faster cooling rate (>10 K/min) was applied. Structural analyses carried out by using single-crystal (Form A) and powder (Form B) X-ray diffraction revealed a rotational disorder of anionic octahedrons and, more interestingly, large conformational differences between cationic moieties caused by their molecular flexibility. Crystal growth of [pbmim][PF6] (Form A) in methanol often leads to numerous crystal defects and revealed that most of them consist of liquid inclusions. The supersaturation ratio (β) appeared to be the predominant factor influencing the crystal growth behavior under isothermal and stagnant conditions. At low β values, a morphological transition from rod-shaped crystals to platelets was observed, presumably caused by a change in the growth mechanism of specific faces. Using high β values promotes either the formation of microscopic (<5 μm) liquid inclusions that become easily detectable upon heating or the appearance of macroscopic inclusions with an hourglass shape.

Molecular dynamics simulations of water permeation across Nafion membrane interfaces.

Permeation of water across the membrane/vapor and membrane/liquid-water interfaces of Nafion is studied using nonequilibrium molecular dynamics (NEMD) simulations, providing direct calculations of mass-transfer resistance. Water mass transfer within one nanometer of the vapor interface is shown to be 2 orders of magnitude slower than at any other point within the membrane, in qualitative agreement with permeation experiments. This interfacial resistance is much stronger than the resistance suggested by prior simulation work calculating self-diffusivity near the interface. The key difference between the prior approach and the NEMD approach is that the NEMD approach implicitly incorporates changes in solubility in the direction normal to the interface. Water is shown to be very insoluble near the vapor interface, which is rich in hydrophobic perfluorocarbon chains, in agreement with advancing contact angle experiments. Hydrophilic side chains are buried beneath this hydrophobic layer and aligned toward the interior of the membrane. Hydrophilic pores are not exposed to the vapor interface as proposed in prior theoretical work. At the membrane/liquid-water interface, highly swollen polymer chains extend into the liquid-water phase, forming a nanoscopically rough interface that is consistent with atomic force microscopy experiments. In these swollen conformations, hydrophilic side chains are exposed to the liquid-water phase, suggesting that the interface is hydrophilic, in agreement with receding contact angle experiments. The mass-transfer resistance of this interface is negligible compared to that of the bulk, in qualitative agreement with permeation experiments. The water activity at the vapor and liquid-water interfaces are nearly the same, yet large conformational and transport differences are observed, consistent with a mass-transfer-based understanding of Schroeder's paradox for Nafion.

Ruxolitinib Mediated Paradox JAK2 Hyperphosphorylation Is Due To The Protection Of Activation Loop Phosphotyrosines From Phosphatases

The JAK2 V617F mutation has been reported in the majority of bcr-abl negative myeloproliferative neoplasms (MPNs) including polycythemia vera (PV:90%), essential thrombocythemia (ET:50%) and idiopathic myelofibrosis (IMF:50%). Recently, ruxolitinib (Jakavi, INCB018424) a JAK2/JAK1 specific inhibitor has been approved by the FDA for the treatment of myelofibrosis. Unexpectedly, treatment of V617FJAK2 expressing cells with ruxolitinib causes paradoxical hyperphosphorylation of JAK2 at its two critical sites (tyr1007/tyr1008). The mechanism of ruxolitinib induced JAK2 hyperphosphorylation is not understood. It has been demonstrated that also the pseudokinase domain of JAK2 display kinase activity and plays a major role in regulating the kinase activity of JAK2. Deletion of the pseudokinase domain leads to hyperphosphorylation of JAK2. We thus hypothesized that ATP-competitive inhibitors might target the kinase activity of the pseudokinase domain. However, treatment of kinase dead pseudokinase domain JAK2V617F mutants with ruxolitinib also causes JAK2 hyperphosphorylation indicating that ruxolitinib mediated JAK2 hyperphosphorylation is not due to inhibition of the pseudokinase domain. We then reasoned that treatment with ruxolitinib might protect JAK2 from tyrosine phosphatases. Co-immunoprecipitation results however did not suggested that ruxolitinib treatment leads to the dissociation of phosphatases such as SHP-2. A Ruxolitinib resistant JAK2V617F mutant (V617F+L902QJAK2) and a JAK2V617F kinase dead mutant (V617F+K882RJAK2) both did not show paradox hyperphoshorylation after ruxolitinib treatment. This indicated that ruxolitinib mediated JAK2 hyperphosphorylation is mediated by a JAK2 intrinsic mechanism. Finally, native-immunocomplexes (non-denaturing IP) of JAK2V617F generated with a monoclonal antibody against ptyr1007/1008 could be blocked by the presence of ruxolitinib, although JAK2V617F was hyperphosphorylated at these sites. We therefore believe now that in the presence of ruxolitinib the JAK2 activation loop folds inside the kinase domain. This leads to the protection of ptyr1007/1008 sites from phosphatases. Most tyrosine kinases are structurally very plastic enzymes with large conformational differences observed between the inactive and active states of the kinase. These conformational changes are largely governed by disorder to order changes in N-lobe, activation loop and hydrophobic motif. When JAK2 is in an inhibited conformational restricted state, the phosphate group of tyr1007 and tyr1008 have an intermolecular interaction with Arg975 and Lys999. These interactions play a major role in the stabilization of the activation loop conformation inside the kinase domain. Mutation of Arg975 and Lys999 to Ala (V617F+R975A), (V617F+L999A) reduced the phosphorylation at both tyr1007/1008 residues. Importantly, ruxolitinib treatment did not lead to JAK2 hyperphosphorylation in this mutants. Taken together our results suggest a novel mode of kinase regulation by modulating kinase activity through conformational changes induced by ruxolutinib. Disclosures:No relevant conflicts of interest to declare.

Distance-independent charge recombination kinetics in cytochrome c-cytochrome c peroxidase complexes: compensating changes in the electronic coupling and reorganization energies.

Charge recombination rate constants vary no more than 3-fold for interprotein ET in the Zn-substituted wild type (WT) cytochrome c peroxidase (CcP):cytochrome c (Cc) complex and in complexes with four mutants of the Cc protein (i.e., F82S, F82W, F82Y, and F82I), despite large differences in the ET distance. Theoretical analysis indicates that charge recombination for all complexes involves a combination of tunneling and hopping via Trp191. For three of the five structures (WT and F82S(W)), the protein favors hopping more than that in the other two structures that have longer heme → ZnP distances (F82Y(I)). Experimentally observed biexponential ET kinetics is explained by the complex locking in alternative coupling pathways, where the acceptor hole state is either primarily localized on ZnP (slow phase) or on Trp191 (fast phase). The large conformational differences between the CcP:Cc interface for the F82Y(I) mutants compared to that the WT and F82S(W) complexes are predicted to change the reorganization energies for the CcP:Cc ET reactions because of changes in solvent exposure and interprotein ET distances. Since the recombination reaction is likely to occur in the inverted Marcus regime, an increased reorganization energy compensates the decreased role for hopping recombination (and the longer transfer distance) in the F82Y(I) mutants. Taken together, coupling pathway and reorganization energy effects for the five protein complexes explain the observed insensitivity of recombination kinetics to donor-acceptor distance and docking pose and also reveals how hopping through aromatic residues can accelerate long-range ET.

Dissecting the in vivo assembly of the 30S ribosomal subunit reveals the role of RimM and general features of the assembly process

Ribosome biogenesis is a tightly regulated, multi-stepped process. The assembly of ribosomal subunits is a central step of the complex biogenesis process, involving nearly 30 protein factors in vivo in bacteria. Although the assembly process has been extensively studied in vitro for over 40 years, very limited information is known for the in vivo process and specific roles of assembly factors. Such an example is ribosome maturation factor M (RimM), a factor involved in the late-stage assembly of the 30S subunit. Here, we combined quantitative mass spectrometry and cryo-electron microscopy to characterize the in vivo 30S assembly intermediates isolated from mutant Escherichia coli strains with genes for assembly factors deleted. Our compositional and structural data show that the assembly of the 3′-domain of the 30S subunit is severely delayed in these intermediates, featured with highly underrepresented 3′-domain proteins and large conformational difference compared with the mature 30S subunit. Further analysis indicates that RimM functions not only to promote the assembly of a few 3′-domain proteins but also to stabilize the rRNA tertiary structure. More importantly, this study reveals intriguing similarities and dissimilarities between the in vitro and the in vivo assembly pathways, suggesting that they are in general similar but with subtle differences.

Crystallographic and NMR Evidence for Flexibility in Oligosaccharyltransferases and Its Catalytic Significance

Oligosaccharyltransferase (OST) is a membrane-bound enzyme that catalyzes the transfer of an oligosaccharide to an asparagine residue in glycoproteins. It possesses a binding pocket that recognizes Ser and Thr residues at the+2 position in theN-glycosylation consensus, Asn-X-Ser/Thr. We determined the crystal structures of the C-terminal globular domains of the catalytic subunits of two archaeal OSTs. A comparison with previously determined structures identified a segment with remarkable conformational plasticity, induced by crystal contact effects. We characterized its dynamic properties in solution by (15)N NMR relaxation analyses. Intriguingly, the mobile region contains the+2 Ser/Thr-binding pocket. In agreement, the flexibility restriction forced by an engineered disulfide crosslink abolished the enzymatic activity, and its cleavage fully restored activity. These results suggest the necessity of multiple conformational states in the reaction. The dynamic nature of the Ser/Thr pocket could facilitate the efficient scanning of N-glycosylation sequons along nascent polypeptide chains.

Overcoming sequence misalignments with weighted structural superposition

An appropriate structural superposition identifies similarities and differences between homologous proteins that are not evident from sequence alignments alone. We have coupled our Gaussian-weighted RMSD (wRMSD) tool with a sequence aligner and seed extension (SE) algorithm to create a robust technique for overlaying structures and aligning sequences of homologous proteins (HwRMSD). HwRMSD overcomes errors in the initial sequence alignment that would normally propagate into a standard RMSD overlay. SE can generate a corrected sequence alignment from the improved structural superposition obtained by wRMSD. HwRMSD's robust performance and its superiority over standard RMSD are demonstrated over a range of homologous proteins. Its better overlay results in corrected sequence alignments with good agreement to HOMSTRAD. Finally, HwRMSD is compared to established structural alignment methods: FATCAT, secondary-structure matching, combinatorial extension, and Dalilite. Most methods are comparable at placing residue pairs within 2 Å, but HwRMSD places many more residue pairs within 1 Å, providing a clear advantage. Such high accuracy is essential in drug design, where small distances can have a large impact on computational predictions. This level of accuracy is also needed to correct sequence alignments in an automated fashion, especially for omics-scale analysis. HwRMSD can align homologs with low-sequence identity and large conformational differences, cases where both sequence-based and structural-based methods may fail. The HwRMSD pipeline overcomes the dependency of structural overlays on initial sequence pairing and removes the need to determine the best sequence-alignment method, substitution matrix, and gap parameters for each unique pair of homologs.

Rationalizing the molecular origins of Ru- and Fe-based dyes for dye-sensitized solar cells

As part of an effort to design more efficient dyes for dye-sensitized solar cells (DSCs), structure-property relationships are established in the world's best-performing chemical series of dyes: 2,2'-bipyridyl-4,4'-carboxylatoruthenium(II) complexes. Statistical analysis, based on crystallographic data from the Cambridge Structural Database, is used to determine common structural features and the effects of structural change to its salient molecular constituents. Also included is the report of two new crystal structures for tris(2,2'-bipyridyl)dichlororuthenium(II)hexahydrate and tris(2,2'-bipyridyl)iron(II)dithiocyanate; these add to this statistical enquiry. Results show that the metal (M) core exhibits a distorted octahedral environment with M-N π-backbonding effects affording the propensity of the metal ion towards oxidation. The same characteristics are observed in iron-based analogues. The role of carboxylic groups in this series of dyes is assessed by comparing complexes which contain or are devoid of COOH groups. Space-group variation and large molecular conformational differences occur when COOH groups are present, while such structural features are very similar in their absence. The nature of the anion is also shown to influence the structure of COOH-containing complexes. These structural findings are corroborated by solution-based UV-vis absorption spectroscopy and DSC device performance tests. The presence of COOH groups in this series of compounds is shown to be mandatory for dye-uptake in TiO(2) in the DSC fabrication process. Throughout this study, results are compared with those of the world's most famous DSC dye, N3 (N719 in its fully protonated form): cis-bis(isothiocyanato)bis(2,2'-bipyridyl-4,4'-dicarboxylato)ruthenium(II). Overall, the molecular origins of charge-transfer in these complexes are ascertained. The findings have important implications to the materials discovery of more efficient dyes for DSC technology.

Structural mechanism of the ATP-induced dissociation of rigor myosin from actin

Myosin is a true nanomachine, which produces mechanical force from ATP hydrolysis by cyclically interacting with actin filaments in a four-step cycle. The principle underlying each step is that structural changes in separate regions of the protein must be mechanically coupled. The step in which myosin dissociates from tightly bound actin (the rigor state) is triggered by the 30 Å distant binding of ATP. Large conformational differences between the crystal structures make it difficult to perceive the coupling mechanism. Energetically accessible transition pathways computed at atomic detail reveal a simple coupling mechanism for the reciprocal binding of ATP and actin.

Species-Specific and Inhibitor-Dependent Conformations of LpxC: Implications for Antibiotic Design

LpxC is an essential enzyme in the lipid A biosynthetic pathway in gram-negative bacteria. Several promising antimicrobial lead compounds targeting LpxC have been reported, though they typically display a large variation in potency against different gram-negative pathogens. We report that inhibitors with a diacetylene scaffold effectively overcome the resistance caused by sequence variation in the LpxC substrate-binding passage. Compound binding is captured in complex with representative LpxC orthologs, and structural analysis reveals large conformational differences that mostly reflect inherent molecular features of distinct LpxC orthologs, whereas ligand-induced structural adaptations occur at a smaller scale. These observations highlight the need for a molecular understanding of inherent structural features and conformational plasticity of LpxC enzymes for optimizing LpxC inhibitors as broad-spectrum antibiotics against gram-negative infections.

Apo and Nickel-Bound Forms of the Pyrococcus horikoshii Species of the Metalloregulatory Protein: NikR Characterized by Molecular Dynamics Simulations

NikR is a homotetrameric nickel regulatory protein whose binding to free Ni(2+) increases its binding affinity for a gene that codes for a nickel transporter protein. It is comprised of a tetrameric nickel-binding domain, flanked by two dimeric DNA-binding domains. Though X-ray crystallography data for various species (Escherichia coli, Heliobacter pylori, and Pyrococcus horikoshii) of NikR reveal large conformational differences between nickel-bound, DNA-bound, and unbound forms, transitions between them have never been observed. We have run all-atom molecular dynamics simulations of three forms of the Pyrococcus horikoshii species of NikR including two apo-forms and one nickel-bound form. Though all 552 residues of this species occur naturally, quantum-mechanics-based force-field parametrization was required to accurately represent the four nickel-centers in the nickel-bound form. Global conformational analysis of the three 100-ns-long simulations indicates slow conformational kinetics and independent DNA binding domain motion. Correlation and flexibility analysis revealed regions of high structural and dynamical importance. A striking relationship was observed between regions with high levels of structural importance and regions with known biological importance. Mutation of key regions of P. horikoshii and analogous regions in both E. coli and H. pylori are suggested that might inhibit DNA-binding activity while not affecting nickel-binding.

Transmembrane Structures of Amyloid Precursor Protein Dimer Predicted by Replica-Exchange Molecular Dynamics Simulations

Abeta peptide is an essential protein in the pathogenesis of Alzheimer's disease and is derived from amyloid precursor protein (APP) in the membrane by beta- and gamma-secretase cleavage. An experimental study has shown that a pairwise replacement of Gly with Leu in APP enhances homodimerization but leads to a drastic reduction of Abeta secretion. To resolve this apparent discrepancy, we predicted the wild-type (WT) and mutant APP dimer conformations by replica-exchange molecular dynamics simulations using the implicit membrane model IMM1. The simulations illustrate large conformational differences between the WT and mutant APP fragments in the membrane. Dimerization of the WT is due to two C(alpha)-H...O hydrogen bonds between two APP fragments, whereas dimerization of the mutant is due to hydrophobic interactions. In the mutant, each APP fragment is more tilted, and the gamma-cleavage site is shifted toward the center of the membrane. This position produces a mismatch between the active site of gamma-secretase and the gamma-cleavage site of APP that might prohibit Abeta production.

Iron-mediated Aggregation and a Localized Structural Change Characterize Ferritin from a Mutant Light Chain Polypeptide That Causes Neurodegeneration

Nucleotide insertions in the ferritin light chain (FTL) polypeptide gene cause hereditary ferritinopathy, a neurodegenerative disease characterized by abnormal accumulation of ferritin and iron in the central nervous system. Here we describe for the first time the protein structure and iron storage function of the FTL mutant p.Phe167SerfsX26 (MT-FTL), which has a C terminus altered in sequence and extended in length. MT-FTL polypeptides assembled spontaneously into soluble, spherical 24-mers that were ultrastructurally indistinguishable from those of the wild type. Far-UV CD showed a decrease in α-helical content, and 8-anilino-1-naphthalenesulfonate fluorescence revealed the appearance of hydrophobic binding sites. Near-UV CD and proteolysis studies suggested little or no structural alteration outside of the C-terminal region. In contrast to wild type, MT-FTL homopolymers precipitated at much lower iron loading, had a diminished capacity to incorporate iron, and were less thermostable. However, precipitation was significantly reversed by addition of iron chelators both in vitro and in vivo. Our results reveal substantial protein conformational changes localized at the 4-fold pore of MT-FTL homopolymers and imply that the C terminus of the MT-FTL polypeptide plays an important role in ferritin solubility, stability, and iron management. We propose that the protrusion of some portion of the C terminus above the spherical shell allows it to cross-link with other mutant polypeptides through iron bridging, leading to enhanced mutant precipitation by iron. Our data suggest that hereditary ferritinopathy pathogenesis is likely to result from a combination of reduction in iron storage function and enhanced toxicity associated with iron-induced ferritin aggregates.