Wild-type Homodimers Research Articles

Mutation S249C of FGFR3b Promotes Bladder Cancer through Downstream Signaling Proteins FRS2 and FRS3: A Computational Approach

Background: Bladder cancer is the 9th most prevalent malignancy worldwide. Fibroblast Growth Factor Receptor 3b (FGFR3b), involved in cell proliferation, differentiation, and migration, is a mutations hotspot for bladder cancer with the most prevalent aberration being S249C. Objective: Impact of S249C of FGFR3b on bladder tumorigenesis via immediate downstream adapter proteins, Fibroblast Growth Factor Receptor Substrate (FRS2 and FRS3) is analyzed computationally. Methods: Wildtype FGFR3b monomer was modeled using I-TASSER and Phyre2. Whereas, S249C mutation was introduced via DynaMut. Wildtype FGFR3b homodimer and mutant heterodimer were structured and docked with downstream proteins using HADDOCK. PDBSum was used to study the amino acid residues involved in intermolecular and intramolecular interactions. Results: Parameters of molecular flexibility and interatomic interactions predicted S249C heterodimer mutation of FGFR3b to be stable. Furthermore, docking with FRS2 protein revealed greater stability and higher binding affinity for S249C heterodimer mutant compared to wildtype homodimer. However, FRS3 docking showed a negligible decline in binding affinity for the S249C mutation but based on Van der Waal’s energy and insights into the interacting residues, it was revealed that these interactions might be stronger and for longer duration in comparison to the wildtype homodimer. Conclusion: S249C heterodimer mutation of FGFR3b is predicted to be stable with a tumorigenic potential where FRS2 and FRS3 might be among the key players of altered downstream signaling. Further investigations are required for a detailed picture.

A focus on dominant negative variants in a series of 170 heterozygous FXI-deficient patients.

Dominant-negative effects have been described for 10F11 variants in the literature. The current study aimed at identifying putative dominant-negative F11 variants. This research consisted in a retrospective analysis of routine laboratory data. In a series of 170patients with moderate/mild factorXI (FXI) deficiencies, we identified heterozygous carriers of previously reported dominant-negative variants (p.Ser243Phe, p.Cys416Tyr, and p.Gly418Val) with FXI activities inconsistent with a dominant-negative effect. Our findings also do not support a dominant-negative effect of p.Gly418Ala. We also identified a set of patients carrying heterozygous variants, among which five out of 11 are novel, with FXI activities suggesting a dominant-negative effect (p.His53Tyr, p.Cys110Gly, p.Cys140Tyr, p.Glu245Lys, p.Trp246Cys, p.Glu315Lys, p.Ile421Thr, p.Trp425Cys, p.Glu565Lys, p.Thr593Met, and p.Trp617Ter). However, for all but two of these variants, individuals with close to half normal FXI coagulant activity (FXI:C) were identified, indicating an inconstant dominant effect. Our data show that for some F11 variants recognized has having dominant-negative effects, such effects actually do not occur in many individuals. The present data suggest that for these patients, the intracellular quality control mechanisms eliminate the variant monomeric polypeptide before homodimer assembly, thereby allowing only the wild-type homodimer to assemble and resulting in half normal activities. In contrast, in patients with markedly decreased activities, some mutant polypeptides might escape this first quality control. In turn, assembly of heterodimeric molecules as well as mutant homodimers would result in activities closer to 1:4 of FXI:C normal range.

Characterization of dominant-negative growth hormone receptor variants reveals a potential therapeutic target for short stature.

Growth hormone insensitivity (GHI) encompasses growth restriction, normal/elevated growth hormone (GH), and low insulin-like growth factor I (IGF1). "Nonclassical" GHI is poorly characterized and is rarely caused by heterozygous dominant-negative (DN) variants located in the intracellular or transmembrane domains of the GH receptor (GHR). We sought to determine the molecular mechanisms underpinning the growth restriction in 2 GHI cases. A custom-made genetic investigative pipeline was exploited to identify the genetic cause of growth restriction in patients with GHI. Nanoluc binary technology (NanoBiT), in vitro splicing assays, western blotting, and flow cytometry, characterized the novel GHR variants. Novel heterozygous GHR variants were identified in 2 unrelated patients with GHI. In vitro splicing assays indicated both variants activated the same alternative splice acceptor site resulting in aberrant splicing and exclusion of 26 base pairs of GHR exon 9. The GHR variants produced truncated receptors and impaired GH-induced GHR signaling. NanoBiT complementation and flow cytometry showed increased cell surface expression of variant GHR homo/heterodimers compared to wild-type (WT) homodimers and increased recombinant human GH binding to variant GHR homo/heterodimers and GH binding protein (GHBP) cleaved from the variant GHRs. The findings demonstrated increased variant GHR dimers and GHBP with resultant GH sequestration. We identified and characterized 2 novel, naturally occurring truncated GHR gene variants. Intriguingly, these DN GHR variants act via the same cryptic splice acceptor site, highlighting impairing GH binding to excess GHBP as a potential therapeutic approach.

Effect of Substitution Pro32Thr on the Interaction between Dimer Subunits of Human Phosphatase ITPA

Background: Cells have specific enzymes (nucleoside triphosphate pyrophosphohydrolase) that hydrolyze non-canonic nucleoside triphosphates into nucleoside monophosphophates and pyrophosphate, thus removing them from the metabolic processes. This class of enzymes includes inosine triphosphate pyrophosphatase (ITPA) which has specificity to ITP, dITP, XTP and dXTP. Objective: The mutation (94C→A) rather often occurs in humans and can affect the sensitivity of patients to medicines. This mutation leads to a Pro32Thr substitution in the human ITPA protein. The mechanism for the inactivating effect of the mutation is unknown yet. Methods: Molecular modeling of the polymorphic form of inosine triphosphate pyrophosphohydrolase Р32Т-hITPA showing the greatest decrease in the enzyme activity is performed. The analysis is given for four dimer variants: wild-type (P32/P32) and mutant (T32/T32) homodimers and two mutant heterodimers (Р32/Т32 and Т32/Р32). Results: The analysis does not show the motion of the loop between α2 and β2 where mutation localized. Thus, the hypothesis of the flipped-out hydrophobic residue and subsequent of protein degradation have not been confirmed. Dimer displacements were much higher than subunit displacements. The analysis of hydrogen bonds between subunits shows that there are the more stable hydrogen bonds in the wild-type homodimer and fewer in the mutant homodimer, while heterodimers have intermediate stability. Conclusion: The results confirm the assumption of possible weakening of bonds between the mutant subunits

Kinase activity of mutant LRRK2 manifests differently in hetero-dimeric vs. homo-dimeric complexes.

The Parkinson's disease (PD) protein leucine-rich repeat kinase 2 (LRRK2) exists as a mixture of monomeric and dimeric species, with its kinase activity highly concentrated in the dimeric conformation of the enzyme. We have adapted the proximity biotinylation approach to study the formation and activity of LRRK2 dimers isolated from cultured cells. We find that the R1441C and I2020T mutations both enhance the rate of dimer formation, whereas, the G2019S kinase domain mutant is similar to WT, and the G2385R risk factor variant de-stabilizes dimers. Interestingly, we find a marked departure in the kinase activity between G2019S-LRRK2 homo-dimers and wild-type-G2019S hetero-dimers. While the homo-dimeric G2019S-LRRK2 exhibits the typical robust enhancement of kinase activity, hetero-dimers comprised of wild-type (WT) and G2019S-LRRK2 exhibit kinase activity similar to WT. Dimeric complexes of specific mutant forms of LRRK2 show reduced stability following an in vitro kinase reaction, in LRRK2 mutants for which the kinase activity is similar to WT. Phosphorylation of the small GTPase Rab10 follows a similar pattern in which hetero-dimers of WT and mutant LRRK2 show similar levels of phosphorylation of Rab10 to WT homo-dimers; while the levels of pRab10 are significantly increased in cells expressing mutant homo-dimers. Interestingly, while the risk variant G2385R leads to a de-stabilization of LRRK2 dimers, those dimers possess significantly elevated kinase activity. The vast majority of familial LRRK2-dependent PD cases are heterozygous; thus, these findings raise the possibility that a crucial factor in disease pathogenesis may be the accumulation of homo-dimeric mutant LRRK2.

High-Speed Observations of the Unbinding/Binding Motions of the Leading Head of Kinesin-1 From/To Microtubule Provide Kinetic Evidence for the Front-Head Gating Mechanism

Kinesin-1 is a motor protein that moves processively along microtubule by alternately moving two motor domains (“heads”). To move in the hand-over-hand manner, ATP hydrolysis cycles of the two heads should be coordinated such that the trailing head hydrolyses ATP and detaches from the microtubule before the leading head does. To explain such coordination, the front-head gating model has been proposed that ATP binding or hydrolysis in the leading head is prohibited due to the backward tension posed through the neck linker. However, it has been technically difficult to directly measure the detachment rate of the leading head. Here, we utilized a heterodimer composed of the wild-type head and a E236A mutant head that hydrolyses ATP very slowly. Single molecule FRET observations between dyes attached to two heads showed that the wild-type head spent majority of time in the leading position under saturating ATP condition. Then we attached a 40 nm-ϕ gold particle specifically to the wild-type head and observed its motion using total internal reflection-based dark-field microscopy at 50 μs temporal resolution. At this resolution, we could clearly distinguish microtubule-bound and -unbound states by the difference in the magnitude of fluctuation of the gold probe. The dwell time of the leading head in the microtubule-bound state was 160 ms on average at 1 mM ATP condition, which was significantly larger than the dwell time of a head of wild-type homodimer in the bound state at trailing position (10 ms; Isojima et al. Nat. Chem. Biol. 2016). These results provide kinetic evidence for the front-head gating model.

Swapping Interface Contacts in the Homodimeric tRNA-Guanine Transglycosylase: An Option for Functional Regulation.

The enzyme tRNA-guanine transglycosylase, a target to fight Shigellosis, recognizes tRNA only as a homodimer and performs full nucleobase exchange at the wobble position. Active-site inhibitors block the enzyme function by competitively replacing tRNA. In solution, the wild-type homodimer dissociates only marginally, whereas mutated variants show substantial monomerization in solution. Surprisingly, one inhibitor transforms the protein into a twisted state, whereby one monomer unit rotates by approximately 130°. In this altered geometry, the enzyme is no longer capable of binding and processing tRNA. Three sugar-type inhibitors have been designed and synthesized, which bind to the protein in either the functionally competent or twisted inactive state. They crystallize with the enzyme side-by-side under identical conditions from the same crystallization well. Possibly, the twisted inactive form corresponds to a resting state of the enzyme, important for its functional regulation.

Ligation events influence ALG-2 dimerization

ALG-2 dimerization was studied using Förster resonance-energy-transfer. D162C variants of ALG-2des23 were covalently modified with Alexa Fluor 488 and Alexa Fluor 647. When samples of the two labeled protein-preparations are combined, the sensitized emission from AF647 serves as a sensitive probe of dimer formation. At 25 °C, in the absence of divalent ions, the wild-type homodimer, ΔGF122 homodimer, and heterodimer display dissociation constants of 7.1, 26, and 4.5 nM, respectively. At 35 °C, subunit interaction is weaker, indicating that dimer formation is exothermic. Binding of Mg2+ in the C-terminal EF-hand (EF5) dramatically enhances ALG-2 dimer stability. Although occupation of EF5 by Ca2+ likewise has a stabilizing effect, its direct influence on dimer stability would be negligible at cytosolic Ca2+ levels. However, dimer stability is substantially increased by the Ca2+-dependent binding of ALG-2 target-peptides, suggesting that the occupation-status of the target-protein binding site is communicated to the dimer interface. Tween 20 is commonly used to improve ALG-2 solubility, the micelles ostensibly acting as target-protein surrogates. Paradoxically, however, the detergent markedly destabilizes ALG-2 dimers, particularly in the presence of Ca2+.

Receptor homodimerization plays a critical role in a novel dominant negative P2RY12 variant identified in a family with severe bleeding

Background Although inherited platelet disorders are still underdiagnosed worldwide, advances in molecular techniques are improving disease diagnosis and patient management. Objective To identify and characterize the mechanism underlying the bleeding phenotype in a Caucasian family with an autosomal dominant P2RY12 variant. Methods Full blood counts, platelet aggregometry, flow cytometry and western blotting were performed before next-generation sequencing (NGS). Detailed molecular analysis of the identified variant of the P2Y12 receptor (P2Y12R) was subsequently performed in mammalian cells overexpressing receptor constructs. Results All three referred individuals had markedly impaired ADP-induced platelet aggregation with primary wave only, despite normal total and surface P2Y12R expression. By NGS, a single P2RY12:c.G794C substitution (p.R265P) was identified in all affected individuals, and this was confirmed by Sanger sequencing. Mammalian cell experiments with the R265P-P2Y12R variant showed normal receptor surface expression versus wild-type (WT) P2Y12R. Agonist-stimulated R265P-P2Y12R function (both signaling and surface receptor loss) was reduced versus WT P2Y12R. Critically, R265P-P2Y12R acted in a dominant negative manner, with agonist-stimulated WT P2Y12R activity being reduced by variant coexpression, suggesting dramatic loss of WT homodimers. Importantly, platelet P2RY12 cDNA cloning and sequencing in two affected individuals also revealed three-fold mutant mRNA overexpression, decreasing even further the likelihood of WT homodimer formation. R265 located within extracellular loop 3 (EL3) is one of four residues that are important for receptor functional integrity, maintaining the binding pocket conformation and allowing rotation following ligand binding. Conclusion This novel dominant negative variant confirms the important role of R265 in EL3 in the functional integrity of P2Y12R, and suggests that pathologic heterodimer formation may underlie this family bleeding phenotype.

Alzheimer's Protective Cross-Interaction Between Wild-Type and A2T Variants Changes Aβ42 Dimer Structure

Whole genome sequencing has recently revealed the protective effect of a single A2T mutation in heterozygous carriers against Alzheimer's disease (AD) and age-related cognitive decline. The impact of the protective cross-interaction between the wild-type (WT) and A2T variants on the dimer structure is therefore of high interest, as the Aβ dimers are smallest known neurotoxic species. Toward this goal, extensive atomistic replica exchange molecular dynamics simulations of the solvated WT homo- and A2T hetero- Aβ1-42 dimers have been performed, resulting into a total of 51 μs of sampling for each system. Simulated heterodimer reveals a striking lowering of transient interactions formed between central and C-terminal hydrophobic regions, when compared to the WT homodimer. Interestingly, the A2T N-terminus forms intra- and inter-peptide contacts with the central and C-terminal residues. This atypical involvement of the N-terminus within A2T heterodimer revealed in our simulations implies possible interference on Aβ42 aggregation and toxic oligomer formation, which is consistent with experiments. In conclusion, the present study provides detailed structural insights onto the AD protective effect of the A2T mutation in the heterozygous state.

Half-of-the-Sites Reactivity of the Castor Δ9-18:0-Acyl Carrier Protein Desaturase.

Fatty acid desaturases regulate the unsaturation status of cellular lipids. They comprise two distinct evolutionary lineages, a soluble class found in the plastids of higher plants and an integral membrane class found in plants, yeast (Saccharomyces cerevisiae), animals, and bacteria. Both classes exhibit a dimeric quaternary structure. Here, we test the functional significance of dimeric organization of the soluble castor Δ9-18:0-acyl carrier protein desaturase, specifically, the hypothesis that the enzyme uses an alternating subunit half-of-the-sites reactivity mechanism whereby substrate binding to one subunit is coordinated with product release from the other subunit. Using a fluorescence resonance energy transfer assay, we demonstrated that dimers stably associate at concentrations typical of desaturase assays. An active site mutant T104K/S202E, designed to occlude the substrate binding cavity, was expressed, purified, and its properties validated by x-ray crystallography, size exclusion chromatography, and activity assay. Heterodimers comprising distinctly tagged wild-type and inactive mutant subunits were purified at 1:1 stoichiometry. Despite having only one-half the number of active sites, purified heterodimers exhibit equivalent activity to wild-type homodimers, consistent with half-of-the-sites reactivity. However, because multiple rounds of turnover were observed, we conclude that substrate binding to one subunit is not required to facilitate product release from the second subunit. The observed half-of-the-sites reactivity could potentially buffer desaturase activity from oxidative inactivation. That soluble desaturases require only one active subunit per dimer for full activity represents a mechanistic difference from the membrane class of desaturases such as the Δ9-acyl-CoA, Ole1p, from yeast, which requires two catalytically competent subunits for activity.

Simulation of mutant P32T homo- and heterodimers of human inosine triphosphate pyrophosphatase hITPA

The structure of three forms of a dimeric enzyme, human inosine triphosphate pyrophosphatase, is considered to identify the enzyme conformation changes causing the inactivation effect of a P32T mutation. Analysis of a nanosecond molecular dynamics is performed; the mean square deviations of the atoms between the wild-type and mutant homodimers, and also the heterodimer are calculated. A 3 ns modeling shows a greater displacement of atoms in mutant protomers. During molecular dynamics simulation, the strongest changes are observed in the loop between α2 and β2 (amino acid residues 28-33, an area of the P32T mutation), the loop between β5 and β6, and the C-terminal amino acid residues. The loop between (α2 and β2 has two conformations characterized by different positions of the Phe31 aromatic group. The distance between Cys33 (Cα) and Phe31 (C(z)) for wild-type and mutant protomers was -9 and 5.5 Å, respectively. These conformations were kept constant.

POLG2 disease variants: analyses reveal a dominant negative heterodimer, altered mitochondrial localization and impaired respiratory capacity.

Human mitochondrial DNA (mtDNA) is replicated and repaired by the mtDNA polymerase gamma, polγ. Polγ is composed of three subunits encoded by two nuclear genes: (1) POLG codes for the 140-kilodalton (kDa) catalytic subunit, p140 and (2) POLG2 encodes the ∼110-kDa homodimeric accessory subunit, p55. Specific mutations are associated with POLG- or POLG2-related disorders. During DNA replication the p55 accessory subunit binds to p140 and increases processivity by preventing polγ's dissociation from the template. To date, studies have demonstrated that homodimeric p55 disease variants are deficient in the ability to stimulate p140; however, all patients currently identified with POLG2-related disorders are heterozygotes. In these patients, we expect p55 to occur as 25% wild-type (WT) homodimers, 25% variant homodimers and 50% heterodimers. We report the development of a tandem affinity strategy to isolate p55 heterodimers. The WT/G451E p55 heterodimer impairs polγ function in vitro, demonstrating that the POLG2 c.1352G>A/p.G451E mutation encodes a dominant negative protein. To analyze the subcellular consequence of disease mutations in HEK293 cells, we designed plasmids encoding p55 disease variants tagged with green fluorescent protein (GFP). P205R and L475DfsX2 p55 variants exhibit irregular diffuse mitochondrial fluorescence and unlike WT p55, they fail to form distinct puncta associated with mtDNA nucleoids. Furthermore, homogenous preparations of P205R and L475DfsX2 p55 form aberrant reducible multimers. We predict that abnormal protein folding or aggregation or both contribute to the pathophysiology of these disorders. Examination of mitochondrial bioenergetics in stable cell lines overexpressing GFP-tagged p55 variants revealed impaired mitochondrial reserve capacity.

Persistence Length of Tropomyosin Heterodimers Formed from Wild-Type and Mutant Chains

Mutations in many contractile proteins, including coiled-coil tropomyosin, can lead to hypertrophic cardiomyopathy (HCM) and heart failure. In the case of HCM-linked αα-tropomyosin, affected individuals may be heterozygous for wild-type and mutant protein; hence, heterodimers are likely to form. Indeed, HCM-causing mutant D175N and E180G tropomyosins both readily dimerize with wild-type tropomyosin in vitro and the heterodimers formed then can be purified(see Kalyva et al., 2012). Here, we have studied the material properties of the heterodimers containing these mutant tropomyosins. Electron microscopy was carried out on tropomyosin molecules following adsorption and rotary-shadowing of the molecules on untreated, freshly cleaved mica. Images of gently curved heterodimers showing no obvious kinking are qualitatively similar to ones of wild-type and mutant homodimers previously examined (Li et al., 2010, 2012). The new EMs were skeletonized by cubic interpolation using the MatLab PCHIP program as described in Li et al. (2010) and then persistence length (PL) determined by the tangent correlation method (ibid). PL for the heterodimer containing D175N and wild-type chains (89 nm) was found to be slightly lower than that for homodimeric wild-type molecules (107 nm), but the value for the E180G - wild-type heterodimer (69 nm) was much lower, suggesting that the E180G - wild-type heterodimer is more curved or more flexible than wild-type tropomyosin. These results agree with the lower thermal stability recorded for the E180G heterodimer than for the D175N heterodimer or the wild-type homodimer and may explain the increased phenotypic severity manifested by the E180G than by the D175N mutation.

α-Tropomyosin with a D175N or E180G Mutation in Only One Chain Differs from Tropomyosin with Mutations in Both Chains

α-Tropomyosin (Tm) carrying hypertrophic cardiomyopathy mutation D175N or E180G was expressed in Escherichia coli. We have assembled dimers of two polypeptide chains in vitro that carry one (αα*) or two (α*α*) copies of the mutation. We found that the presence of the mutation has little effect on dimer assembly, thereby predicting that individuals heterozygous for the Tm mutations are likely to express both αα* and α*α* Tm. Depending on the expression level, the heterodimer may be the predominant form in individuals carrying the mutation. Thus, it is important to define differences in the properties of Tm molecules carrying one or two copies of the mutation. We examined the Tm homo- and heterodimer properties: actin affinity, thermal stability, calcium regulation of myosin subfragment 1 binding, and calcium regulation of myofibril force. We report that the properties of the heterodimer may be similar to those of the wild-type homodimer (actin affinity, thermal stability, D175N αα*), similar to those of the mutant homodimer (calcium sensitivity, D175N αα*), intermediate between the two (actin affinity, E180G αα*), or different from both (thermal stability, E180G αα*). Thus, the properties of the homodimer are not a completely reliable guide to the properties of the heterodimer.

Parkinson’s disease-associated mutations in DJ-1 modulate its dimerization in living cells

Mutations in the protein DJ-1 cause recessive forms of early onset familial Parkinson’s disease (PD). To date, most of the causative mutations studied destabilize formation of DJ-1 homodimers, which appears to be closely linked to its normal function in oxidative stress and other cellular processes. Despite the importance of understanding the dimerization dynamics of this protein, this aspect of DJ-1 biology has not previously been directly studied in living cells. Here, we use bimolecular fluorescence complementation to study DJ-1 dimerization and find not only that DJ-1 forms homodimers in living cells but that most PD causative DJ-1 mutations disrupt this process, including the L166P, M26I, L10P, and P158∆ mutations. Interestingly, the E64D mutant form of DJ-1 retains the ability to form homodimers. However, while wild-type DJ-1 dimers are stabilized under oxidative stress conditions, we find that the E64D mutation blocks this stabilization. Furthermore, our data show that the E64D mutation potentiates the formation of aggresomes containing DJ-1. We also observe that while the widely studied L166P mutation prevents DJ-1 from forming homodimers or heterodimers with wild-type protein, the mutant protein is able to partially disrupt formation of wild-type homodimers. In summary, by investigating DJ-1 dimerization in living cells, we have uncovered several novel properties of PD causative mutations in DJ-1, which may ultimately provide novel insight into PD pathogenesis and possible therapeutic options.Electronic supplementary materialThe online version of this article (doi:10.1007/s00109-012-0976-y) contains supplementary material, which is available to authorized users.

Computational modeling of the bHLH domain of the transcription factor TWIST1 and R118C, S144R and K145E mutants.

BackgroundHuman TWIST1 is a highly conserved member of the regulatory basic helix-loop-helix (bHLH) transcription factors. TWIST1 forms homo- or heterodimers with E-box proteins, such as E2A (isoforms E12 and E47), MYOD and HAND2. Haploinsufficiency germ-line mutations of the twist1 gene in humans are the main cause of Saethre-Chotzen syndrome (SCS), which is characterized by limb abnormalities and premature fusion of cranial sutures. Because of the importance of TWIST1 in the regulation of embryonic development and its relationship with SCS, along with the lack of an experimentally solved 3D structure, we performed comparative modeling for the TWIST1 bHLH region arranged into wild-type homodimers and heterodimers with E47. In addition, three mutations that promote DNA binding failure (R118C, S144R and K145E) were studied on the TWIST1 monomer. We also explored the behavior of the mutant forms in aqueous solution using molecular dynamics (MD) simulations, focusing on the structural changes of the wild-type versus mutant dimers.ResultsThe solvent-accessible surface area of the homodimers was smaller on wild-type dimers, which indicates that the cleft between the monomers remained more open on the mutant homodimers. RMSD and RMSF analyses indicated that mutated dimers presented values that were higher than those for the wild-type dimers. For a more careful investigation, the monomer was subdivided into four regions: basic, helix I, loop and helix II. The basic domain presented a higher flexibility in all of the parameters that were analyzed, and the mutant dimer basic domains presented values that were higher than the wild-type dimers. The essential dynamic analysis also indicated a higher collective motion for the basic domain.ConclusionsOur results suggest the mutations studied turned the dimers into more unstable structures with a wider cleft, which may be a reason for the loss of DNA binding capacity observed for in vitro circumstances.

Inter-monomer electron transfer is too slow to compete with monomeric turnover in bc1 complex

The homodimeric bc1 complexes are membrane proteins essential in respiration and photosynthesis. The ~11Å distance between the two bL-hemes of the dimer opens the possibility of electron transfer between them, but contradictory reports make such inter-monomer electron transfer controversial. We have constructed in Rhodobacter sphaeroides a heterodimeric expression system similar to those used before, in which the bc1 complex can be mutated differentially in the two copies of cyt b to test for inter-monomer electron transfer, but found that genetic recombination by cross-over then occurs to produce wild-type homodimer. Selection pressure under photosynthetic growth always favored the homodimer over heterodimeric variants enforcing inter-monomer electron transfer, showing that the latter are not competitive. These results, together with kinetic analysis of myxothiazol titrations, demonstrate that inter-monomer electron transfer does not occur at rates competitive with monomeric turnover. We examine the results from other groups interpreted as demonstrating rapid inter-monomer electron transfer, conclude that similar mechanisms are likely to be in play, and suggest that such claims might need to be re-examined.

A Novel Missense Mutation in FGG (c.944C>A) Encodes for An Amino Acid Change (p.Ala315Asp) in the Gamma Chain of Fibrinogen Causing Hypofibrinogenemia and a Thrombotic Phenotype

Abstract 856Fibrinogen plays a central role in maintaining hemostasis. Disruption of its normal function may lead to hemorrhagic or thrombotic events. Fibrinogen is encoded by three genes, FGA, FGB, and FGG, clustered on chromosome 4q28-q31. Hereditary defects of fibrinogen, although uncommon, can affect the quantity (hypofibrinogenemia and afibrinogenemia) or the quality (dysfibrinogenemia) of the circulating protein. We report a family with two affected individuals (father and daughter) that presented with a mild bleeding predisposition, but also exhibited thrombotic events, i.e. unprovoked deep vein thrombosis in a father and an in utero middle cerebral artery stroke in a daughter. The Clauss fibrinogen levels for the father and daughter were 105 and 98 mg/dL (normal 150–400 mg/dL) and fibrinogen antigen levels were 138 and 152 mg/dL (normal 170–400 mg/dL), respectively. Thrombin times were prolonged in both patients. All other coagulation factors were within normal range and the patients did not carry either the prothrombin G20210A or factor V Leiden mutation.All exons and intron-exon junctions of the three fibrinogen genes were sequenced. The father and daughter were found to be heterozygous for a novel missense mutation in the gamma chain (Ala315Asp) while this mutation was not observed in an unaffected family member (mother of the daughter).Transient transfection experiments using CHO-K1 cells showed that gamma chain with p.Ala315Asp mutation was detected in both cell lysates and supernatant, although in reduced amount as compared with cells transfected with wild-type (WT) FGG cDNA.Structurally, there was no significant difference in fibrin fiber diameter, as measured manually by scanning electron microscopy, between the affected (father and daughter), and the daughter's unaffected mother. Similarly, there was no difference in thrombus height between the three individuals. As expected, hydraulic permeability of platelet rich plasma clots correlated with the fibrinogen levels in each person. Clotting kinetics in plasma of affected family members was consistent with hypofibrinogenemia, but did not suggest abnormal fibrin polymerization. Clot structure and fibrin deposition was further characterized with a microfluidics flow assay, an experimental technique in which platelet and fibrin accumulation is observed in real-time using fluorescent probes in whole blood under shear stress on a collagen/tissue factor micropatterned surface. Interestingly, fibrin density and platelet accumulation, as measured by platelet and fibrin surface area coverage, was highest in the father and daughter, the two individuals with hypofibrinogenemia and thrombotic phenotype.In order to evaluate the possible changes resulting from the Ala315Asp mutation we examined predicted alterations in protein structure, energy, and protein-protein interaction between two fibrinogen molecules via in silico molecular modeling using published protein crystal structure. The molecular modeling showed that alanine for aspartic acid substitution predicted to result in substantial changes in overall conformation of the interaction interface (distal D-domain) between the two fibrinogen molecules due to introduction of a bulkier side chain. Potential energy (energy of minimization) for Ala315Asp mutant homodimer, and for WT-mutant heterodimer were lower than the WT homodimer, predicting a more stable structure from an energetic standpoint. Overall interaction energy (measurement of the energy of association between the two molecules) was lower for Ala315Asp mutant homodimer as compared with WT homodimer or WT-mutant heterodimer, suggesting more favorable interaction for Ala315Asp mutant dimers. Overall, the in silico modeling studies predicted that Ala315Asp mutation may lead to more rapid or stronger interaction between fibrinogen molecules.These data suggest that a novel FGG mutation, c.944C>A, that predicts pAla315Asp change, if expressed and incorporated into fibrinogen molecule in vivo, may result in a more stable or more resistant to lysis clot. Characterizing the association between a specific genetic defect and its phenotypic expression will advance the current understanding of the molecular and biochemical mechanisms of the inherited disorders of fibrinogen, and may contribute to the development of safer therapeutic interventions for the patients with fibrinogen disorders. Disclosures:No relevant conflicts of interest to declare.

Mechanism of activation of human c-KIT kinase by internal tandem duplications of the juxtamembrane domain and point mutations at aspartic acid 816

The proto-oncogene c-KIT receptor has been implicated as an essential component in the activation of leukemic cells. The internal tandem duplication (ITD) of c-KIT has also been identified as a predominant cause of acute myeloid leukemia (AML), although its role in the activation process is still unclear. To investigate the biological mechanisms of c-KIT activation, we generated a c-KIT receptor bearing two different immunological tags, HA and Flag tags. In this study, we demonstrated that the mutant (Mt)-ITD and Asp816 (D816Y) c-KIT receptors spontaneously formed dimers and that these Mt-ITD forms of c-KIT displayed high levels of phosphorylation and increased cellular tyrosine phosphorylation. The amount of wild-type homodimers increased following the addition of the c-KIT ligand, while the level of mutant homodimers was less affected by the addition of the c-KIT ligand. Furthermore, we demonstrated that Mt-ITD and activating point mutations of D816Y induced constitutive activation of c-KIT kinase in the absence of ligand in COS-1 cells. These data suggest a novel mechanism for the regulation of cell growth autonomy. Overall, our study suggests that c-KIT activation might have significant effects on hematopoietic cells and might help to improve our understanding of the pathogenesis of systemic mast cell disease, gastrointestinal stromal tumors and AML and potentially lead to the development of novel therapeutic approaches.