Gln Residues Research Articles

Transglutaminase-Based Chemo-Enzymatic Conjugation Approach Yields Homogeneous Antibody–Drug Conjugates

Most chemical techniques used to produce antibody-drug conjugates (ADCs) result in a heterogeneous mixture of species with variable drug-to-antibody ratios (DAR) which will potentially display different pharmacokinetics, stability, and safety profiles. Here we investigated two strategies to obtain homogeneous ADCs based on site-specific modification of deglycosylated antibodies by microbial transglutaminase (MTGase), which forms isopeptidic bonds between Gln and Lys residues. We have previously shown that MTGase solely recognizes Gln295 within the heavy chain of IgGs as a substrate and can therefore be exploited to generate ADCs with an exact DAR of 2. The first strategy included the direct, one-step attachment of the antimitotic toxin monomethyl auristatin E (MMAE) to the antibody via different spacer entities with a primary amine functionality that is recognized as a substrate by MTGase. The second strategy was a chemo-enzymatic, two-step approach whereby a reactive spacer entity comprising a bio-orthogonal thiol or azide function was attached to the antibody by MTGase and subsequently reacted with a suitable MMAE-derivative. To this aim, we investigated two different chemical approaches, namely, thiol-maleimide and strain-promoted azide-alkyne cycloaddition (SPAAC). Direct enzymatic attachment of MMAE-spacer derivatives at an 80 molar excess of drug yielded heterogeneous ADCs with a DAR of between 1.0 to 1.6. In contrast to this, the chemo-enzymatic approach only required a 2.5 molar excess of toxin to yield homogeneous ADCs with a DAR of 2.0 in the case of SPAAC and 1.8 for the thiol-maleimide approach. As a proof-of-concept, trastuzumab (Herceptin) was armed with the MMAE via the chemo-enzymatic approach using SPAAC and tested in vitro. Trastuzumab-MMAE efficiently killed BT-474 and SK-BR-3 cells with an IC50 of 89.0 pM and 21.7 pM, respectively. Thus, the chemo-enzymatic approach using MTGase is an elegant strategy to form ADCs with a defined DAR of 2. Furthermore, the approach is directly applicable to a broad variety of antibodies as it does not require prior genetic modifications of the antibody sequence.

Site-Specific Derivatization of Avidin Using Microbial Transglutaminase

Avidin conjugates have several important applications in biotechnology and medicine. In this work, we investigated the possibility to produce site-specific derivatives of avidin using microbial transglutaminase (TGase). TGase allows the modification of proteins at the level of Gln or Lys residues using as substrate an alkyl-amine or a Gln-mimicking moiety, respectively. The reaction is site-specific, since Gln and Lys derivatization occurs preferentially at residues embedded in flexible regions of protein substrates. An analysis of the X-ray structure of avidin allowed us to predict Gln126 and Lys127 as potential sites of TGase's attack, because these residues are located in the flexible/unfolded C-terminal region of the protein. Surprisingly, incubation of avidin with TGase in the presence of alkylamine containing substrates (dansylcadaverine, 5-hydroxytryptamine) revealed a very low level of derivatization of the Gln126 residue. Analysis of the TGase reaction on synthetic peptide analogues of the C-terminal portion of avidin indicated that the lack of reactivity of Gln126 was likely due to the fact that this residue is proximal to negatively charged carboxylate groups, thus hampering the interaction of the substrate at the negatively charged active site of TGase. On the other hand, incubation of avidin with TGase in the presence of carbobenzoxy-l-glutaminyl-glycine in order to derivatize Lys residue(s) resulted in a clean and high yield production of an avidin derivative, retaining the biotin binding properties and the quaternary structure of the native protein. Proteolytic digestion of the modified protein, followed by mass spectrometry, allowed us to identify Lys127 as the major site of reaction, together with a minor modification of Lys58. By using TGase, avidin was also conjugated via a Lys-Gln isopeptide bond to a protein containing a single reactive Gln residue, namely, Gln126 of granulocyte-macrophage colony-stimulating factor. TGase can thus be exploited for the site-specific derivatization of avidin with small molecules or proteins.

Yet more intramolecular cross-links in Gram-positive surface proteins

The surface of Gram-positive bacteria comprises a single membrane and, typically, a thick layer of cross-linked peptidoglycan that imparts strength and rigidity. Anchored to this cell wall are protein assemblies, such as pili, and surface proteins, such as microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) that act as surface adhesins. These proteins are critical not only for bacterial binding to host surfaces in the early stages of infection but also for biofilm formation and immune evasion. In a series of studies published since 2007, these cell surface proteins and protein assemblies have been shown to contain isopeptide and thioester bonds, highly unusual intramolecular covalent linkages between amino acid side chains. These cross-links have either a structure-stabilizing role or may be directly involved in adhesion. Using an elegant combination of structural biology and mass spectrometry, Kwon et al. (1) reveal in PNAS an ester bond as yet another covalent linkage in a putative MSCRAMM from the Gram-positive pathogen Clostridium perfringens . Remarkably, this ester bond, joining the side chains of a Thr and Gln residue, is equivalent to an unresolved acyl-enzyme intermediate, formed on an autocatalytic pathway that resembles the mechanism of serine proteases. Despite fundamental differences in the way they are encoded in bacterial genomes, following delivery/assembly at the cell surface, pili and many MSCRAMMs share important similarities. In such proteins, a series of repetitive domains (commonly referred to as stalk regions) are responsible for the length of the structure and allow an N-terminal region to be positioned away from the cell surface. At the C terminus, both pili and MSCRAMMs are anchored to the cell wall by sortases (2). Although many of the building blocks of Gram-positive surface proteins and pili bear some resemblance to their Gram-negative … [↵][1]1To whom correspondence should be addressed. E-mail: Mark.Banfield{at}jic.ac.uk. [1]: #xref-corresp-1-1

Difference FT-IR Studies on the Effects of Buffers on Nucleotide Binding to Reca

The Escherichia coli protein RecA catalyzes DNA strand exchange and plays a role DNA repair and genetic recombination. Nucleotide binding influences RecA oligomerization and its affinity for DNA. Previous studies in our lab have shown buffer-specific changes in RecA stability and unfolding transitions. Past circular dichroism (CD), infrared (IR), and fluorescence studies suggest only minimal buffer dependent changes in nucleotide binding and secondary structure that did not explain the large buffer dependent differences in RecA stability and unfolding profiles. These observations led to further investigations of how the four common biological buffers Tris, HEPES, MES, and PO4 alter RecA structure and nucleotide binding. Here we have employed difference infrared spectroscopy utilized in conjunction with caged nucleotides to generate RecA-ADP minus RecA difference infrared spectra in each of the four buffers. These higher resolution studies are aimed at detecting if the buffers alter nucleotide binding to RecA. Preliminary results show that ADP binding results in perturbations in Gln, Glu, Asp, Asn, Tyr, and Lys residues and secondary structural changes. Initial comparisons of difference spectra obtained in the four buffers show some similar changes but also show some differences. These differences between RecA-ADP minus RecA difference spectra will be discussed.

Site-specific protein labelling and immobilization mediated by microbial transglutaminase.

Microbial transglutaminase (mTG) shows broad substrate specificity that is amenable to in vitro bio-conjugation applications. Herein, test proteins were genetically fused with peptide tags, followed by mTG-mediated propargylation of their reactive Gln residues. The propargylated proteins were subjected to copper-assisted azide-alkyne cycloaddition to demonstrate either fluorescent labelling or immobilization.

Functional Characterization of ECP-Heparin Interaction: A Novel Molecular Model

Human eosinophil cationic protein (ECP) and eosinophil derived neurotoxin (EDN) are two ribonuclease A (RNaseA) family members secreted by activated eosinophils. They share conserved catalytic triad and similar three dimensional structures. ECP and EDN are heparin binding proteins with diverse biological functions. We predicted a novel molecular model for ECP binding of heparin hexasaccharide (Hep6), [GlcNS(6S)-IdoA(2S)]3, and residues Gln40, His64 and Arg105 were indicated as major contributions for the interaction. Interestingly, Gln40 and His64 on ECP formed a clamp-like structure to stabilize Hep6 in our model, which was not observed in the corresponding residues on EDN. To validate our prediction, mutant ECPs including ECP Q40A, H64A, R105A, and double mutant ECP Q40A/H64A were generated, and their binding affinity for heparins were measured by isothermal titration calorimetry (ITC). Weaker binding of ECP Q40A/H64A of all heparin variants suggested that Gln40-His64 clamp contributed to ECP-heparin interaction significantly. Our in silico and in vitro data together demonstrate that ECP uses not only major heparin binding region but also use other surrounding residues to interact with heparin. Such correlation in sequence, structure, and function is a unique feature of only higher primate ECP, but not EDN.

Discovery of a linoleate 9S-dioxygenase and an allene oxide synthase in a fusion protein of Fusarium oxysporum

Fusarium oxysporum is a devastating plant pathogen that oxidizes C₁₈ fatty acids sequentially to jasmonates. The genome codes for putative dioxygenase (DOX)-cytochrome P450 (CYP) fusion proteins homologous to linoleate diol synthases (LDSs) and the allene oxide synthase (AOS) of Aspergillus terreus, e.g., FOXB_01332. Recombinant FOXB_01332 oxidized 18:2n-6 to 9S-hydroperoxy-10(E),12(Z)-octadecadienoic acid by hydrogen abstraction and antarafacial insertion of molecular oxygen and sequentially to an allene oxide, 9S(10)-epoxy-10,12(Z)-octadecadienoic acid, as judged from nonenzymatic hydrolysis products (α- and γ-ketols). The enzyme was therefore designated 9S-DOX-AOS. The 9S-DOX activity oxidized C₁₈ and C₂₀ fatty acids of the n-6 and n-3 series to hydroperoxides at the n-9 and n-7 positions, and the n-9 hydroperoxides could be sequentially transformed to allene oxides with only a few exceptions. The AOS activity was stereospecific for 9- and 11-hydroperoxides with S configurations. FOXB_01332 has acidic and alcoholic residues, Glu⁹⁴⁶-Val-Leu-Ser⁹⁴⁹, at positions of crucial Asn and Gln residues (Asn-Xaa-Xaa-Gln) of the AOS and LDS. Site-directed mutagenesis studies revealed that FOXB_01332 and AOS of A. terreus differ in catalytically important residues suggesting that AOS of A. terreus and F. oxysporum belong to different subfamilies. FOXB_01332 is the first linoleate 9-DOX with homology to animal heme peroxidases and the first 9-DOX-AOS fusion protein.

Transmembrane Domain II of the Human Bile Acid Transporter SLC10A2 Coordinates Sodium Translocation

Human apical sodium-dependent bile acid transporter (hASBT, SLC10A2) is responsible for intestinal reabsorption of bile acids and plays a key role in cholesterol homeostasis. We used a targeted and systematic approach to delineate the role of highly conserved transmembrane helix 2 on the expression and function of hASBT. Cysteine mutation significantly depressed transport activity for >60% of mutants without affecting cell surface localization of the transporter. All mutants were inaccessible toward chemical modification by membrane-impermeant MTSET reagent, strongly suggesting that transmembrane 2 (TM2) plays an indirect role in bile acid substrate translocation. Both bile acid uptake and sodium dependence of TM2 mutants revealed a distinct α-helical periodicity. Kinetic studies with conservative and non-conservative mutants of sodium sensitive residues further underscored the importance of Gln(75), Phe(76), Met(79), Gly(83), Leu(86), Phe(90), and Asp(91) in hASBT function. Computational analysis indicated that Asp(91) may coordinate with sodium during the transport cycle. Combined, our data propose that a consortium of sodium-sensitive residues along with previously reported residues (Thr(134), Leu(138), and Thr(149)) from TM3 may form the sodium binding and translocation pathway. Notably, residues Gln(75), Met(79), Thr(82), and Leu(86) from TM2 are highly conserved in TM3 of a putative remote bacterial homologue (ASBTNM), suggesting a universal mechanism for the SLC10A transporter family.

Top‐down HPLC–ESI‐MS characterization of rat gliadoralin A, a new member of the family of rat submandibular gland glutamine‐rich proteins and potential substrate of transglutaminase

During HPLC-ESI-MS/MS analysis of rat submandibular saliva secreted under isoprenaline stimulation, a protein with an experimental [M+H](1+) = 10,544.24 m/z was detected (17.5 ± 0.7 min). The MS/MS fragmentation pattern, manually investigated, allowed establishing an internal sequence in agreement with a DNA-derived sequence of an unknown rat protein coded D3Z9M3 (Swiss-Prot). To match the experimental MS/MS fragmentation pattern and protein mass with theoretical data, the removal from the N terminus of the signal peptide and from the C terminus of three amino acid (a.a.) residues (Arg-Ala-Val) and the cyclization of the N-terminal glutamine in pyroglutamic had to be supposed, resulting in a mature protein of 90 a.a. HPLC-ESI-MS/MS of the trypsin digest ensured 100% sequence coverage. For the high glutamine content (34/90 = 37.8%) we propose to name this protein rat gliadoralin A 1-90. Low amounts of five different isoforms were sporadically detected, which did not significantly change their relative amounts after stimulation. Gliadoralin A is substrate for transglutaminase-2, having Lys 60 and different Gln residues as major determinants for enzyme recognition. In silico investigation of superior structures evidenced that a small part of the protein adopts an α-helical fold, whereas large segments are unfolded, suggesting an unordered conformation.

Molecular Determinants of the Substrate Specificity of the Complement-initiating Protease, C1r

The serine protease, C1r, initiates activation of the classical pathway of complement, which is a crucial innate defense mechanism against pathogens and altered-self cells. C1r both autoactivates and subsequently cleaves and activates C1s. Because complement is implicated in many inflammatory diseases, an understanding of the interaction between C1r and its target substrates is required for the design of effective inhibitors of complement activation. Examination of the active site specificity of C1r using phage library technology revealed clear specificity for Gln at P2 and Ile at P1', which are found in these positions in physiological substrates of C1r. Removal of one or both of the Gln at P2 and Ile at P1' in the C1s substrate reduced the rate of C1r activation. Substituting a Gln residue into the P2 of the activation site of MASP-3, a protein with similar domain structure to C1s that is not normally cleaved by C1r, enabled efficient activation of this enzyme. Molecular dynamics simulations and structural modeling of the interaction of the C1s activation peptide with the active site of C1r revealed the molecular mechanisms that particularly underpin the specificity of the enzyme for the P2 Gln residue. The complement control protein domains of C1r also made important contributions to efficient activation of C1s by this enzyme, indicating that exosite interactions were also important. These data show that C1r specificity is well suited to its cleavage targets and that efficient cleavage of C1s is achieved through both active site and exosite contributions.

Structural organization of FtsB, a transmembrane protein of the bacterial divisome.

We report the first structural analysis of an integral membrane protein of the bacterial divisome. FtsB is a single-pass membrane protein with a periplasmic coiled coil. Its heterologous association with its partner FtsL represents an essential event for the recruitment of the late components to the division site. Using a combination of mutagenesis, computational modeling, and X-ray crystallography, we determined that FtsB self-associates, and we investigated its structural organization. We found that the transmembrane domain of FtsB homo-oligomerizes through an evolutionarily conserved interaction interface where a polar residue (Gln 16) plays a critical role through the formation of an interhelical hydrogen bond. The crystal structure of the periplasmic domain, solved as a fusion with Gp7, shows that 30 juxta-membrane amino acids of FtsB form a canonical coiled coil. The presence of conserved Gly residue in the linker region suggests that flexibility between the transmembrane and coiled coil domains is functionally important. We hypothesize that the transmembrane helices of FtsB form a stable dimeric core for its association with FtsL into a higher-order oligomer and that FtsL is required to stabilize the periplasmic domain of FtsB, leading to the formation of a complex that is competent for binding to FtsQ, and to their consequent recruitment to the divisome. The study provides an experimentally validated structural model and identifies point mutations that disrupt association, thereby establishing important groundwork for the functional characterization of FtsB in vivo.

Expression of Fusion Proteins of Aspergillus terreus Reveals a Novel Allene Oxide Synthase

Aspergilli oxidize C18 unsaturated fatty acids by dioxygenase-cytochrome P450 fusion proteins to signal molecules involved in reproduction and host-pathogen interactions. Aspergillus terreus expresses linoleate 9R-dioxygenase (9R-DOX) and allene oxide synthase (AOS) activities in membrane fractions. The genome contains five genes (ATEG), which may code for a 9R-DOX-AOS fusion protein. The genes were cloned and expressed, but none of them oxidized 18:2n-6 to 9R-hydroperoxy-10(E),12(Z)-octadecadienoic acid (9R-HPODE). ATEG_02036 transformed 9R-HPODE to an unstable allene oxide, 9(R),10-epoxy-10,12(Z)-octadecadienoic acid. A substitution in the P450 domain (C1073S) abolished AOS activity. The N964V and N964D mutants both showed markedly reduced AOS activity, suggesting that Asn(964) may facilitate homolytic cleavage of the dioxygen bond of 9R-HPODE with formation of compound II in analogy with plant AOS (CYP74) and prostacyclin synthase (CYP8A1). ATEG_03992 was identified as 5,8-linoleate diol synthase (5,8-LDS). Replacement of Asn(878) in 5,8-LDS with leucine (N878L) mainly shifted ferryl oxygen insertion from C-5 toward C-6, but replacements of Gln(881) markedly affected catalysis. The Q881L mutant virtually abolished the diol synthase activity. Replacement of Gln(881) with Asn, Glu, Asp, or Lys residues augmented the homolytic cleavage of 8R-HPODE with formation of 10-hydroxy-8(9)-epoxy-12(Z)-octadecenoic acid (erythro/threo, 1-4:1) and/or shifted ferryl oxygen insertion from C-5 toward C-11. We conclude that homolysis and heterolysis of the dioxygen bond with formation of compound II in AOS and compound I in 5,8-LDS are influenced by Asn and Gln residues, respectively, of the I-helices. AOS of A. terreus appears to have evolved independently of CYP74 but with an analogous reaction mechanism.

Molecular basis of peptide activation of the GLP-1 receptor

Molecular basis of peptide activation of the GLP-1 receptor

Geometric and electronic structures of manganese-substituted iron superoxide dismutase.

The active-site structures of the oxidized and reduced forms of manganese-substituted iron superoxide dismutase (Mn(Fe)SOD) are examined, for the first time, using a combination of spectroscopic and computational methods. On the basis of electronic absorption, circular dichroism (CD), magnetic CD (MCD), and variable-temperature variable-field MCD data obtained for oxidized Mn(Fe)SOD, we propose that the active site of this species is virtually identical to that of wild-type manganese SOD (MnSOD), with both containing a metal ion that resides in a trigonal bipyramidal ligand environment. This proposal is corroborated by quantum mechanical/molecular mechanical (QM/MM) computations performed on complete protein models of Mn(Fe)SOD in both its oxidized and reduced states and, for comparison, wild-type (WT) MnSOD. The major differences between the QM/MM optimized active sites of WT MnSOD and Mn(Fe)SOD are a smaller (His)N-Mn-N(His) equatorial angle and a longer (Gln146(69))NH···O(sol) H-bond distance in the metal-substituted protein. Importantly, these modest geometric differences are consistent with our spectroscopic data obtained for the oxidized proteins and high-field electron paramagnetic resonance spectra reported previously for reduced Mn(Fe)SOD and MnSOD. As Mn(Fe)SOD exhibits a reduction midpoint potential (E(m)) almost 700 mV higher than that of MnSOD, which has been shown to be sufficient for explaining the lack of SOD activity displayed by the metal-subtituted species (Vance, C. K.; Miller, A. F. Biochemistry 2001, 40, 13079-13087), E(m)'s were computed for our experimentally validated QM/MM optimized models of Mn(Fe)SOD and MnSOD. These computations properly reproduce the experimental trend and reveal that the drastically elevated E(m) of the metal substituted protein stems from a larger separation between the second-sphere Gln residue and the coordinated solvent in Mn(Fe)SOD relative to MnSOD, which causes a weakening of the corresponding H-bond interaction in the oxidized state and alleviates steric crowding in the reduced state.

Characterization of singlet oxygen production and its involvement in photodamage of Photosystem II in the cyanobacterium Synechocystis PCC 6803 by histidine-mediated chemical trapping

Singlet oxygen production in intact cells of the cynobacterium Synechocystis 6803 was studied using chemical trapping by histidine, which leads to O2 uptake during illumination. The rate of O2 uptake, measured by a standard Clark-type electrode, is enhanced in the presence of D2O, which increases the lifetime of 1O2, and suppressed by the 1O2 quencher NaN3. Due to the limited mobility of 1O2 these data demonstrate that exogenous histidine reaches close vicinity of 1O2 production sites inside the cells. Flash induced chlorophyll fluorescence measurements showed that histidine does not inhibit Photosystem II activity up to 5mM concentration. By applying the histidine-mediated O2 uptake method we showed that 1O2 production linearly increases with light intensity even above the saturation of photosynthesis. We also studied 1O2 production in site directed mutants in which the Gln residue at the 130th position of the D1 reaction center subunit was changed to either Glu or Leu, which affect the efficiency of nonradiative charge recombination from the primary radical pair (Rappaport et al. 2002, Biochemistry 41: 8518–8527; Cser and Vass 2007, BBA 1767:233–243). We found that the D1-Gln130Glu mutant showed decreased 1O2 production concomitant with decreased rate of photodamage relative to the WT, whereas both 1O2 production and photodamage were enhanced in the D1-Gln130Leu mutant. The data are discussed in the framework of the model of photoinhibition in which 3P680 mediated 1O2 production plays a key role in PSII photodamage, and nonradiative charge recombination of the primary charge separated state provides a photoprotective pathway.

Quantitative Characterization of the Activation Steps of Mannan-binding Lectin (MBL)-associated Serine Proteases (MASPs) Points to the Central Role of MASP-1 in the Initiation of the Complement Lectin Pathway

Mannan-binding lectin (MBL)-associated serine proteases, MASP-1 and MASP-2, have been thought to autoactivate when MBL/ficolin·MASP complexes bind to pathogens triggering the complement lectin pathway. Autoactivation of MASPs occurs in two steps: 1) zymogen autoactivation, when one proenzyme cleaves another proenzyme molecule of the same protease, and 2) autocatalytic activation, when the activated protease cleaves its own zymogen. Using recombinant catalytic fragments, we demonstrated that a stable proenzyme MASP-1 variant (R448Q) cleaved the inactive, catalytic site Ser-to-Ala variant (S646A). The autoactivation steps of MASP-1 were separately quantified using these mutants and the wild type enzyme. Analogous mutants were made for MASP-2, and rate constants of the autoactivation steps as well as the possible cross-activation steps between MASP-1 and MASP-2 were determined. Based on the rate constants, a kinetic model of lectin pathway activation was outlined. The zymogen autoactivation rate of MASP-1 is ∼3000-fold higher, and the autocatalytic activation of MASP-1 is about 140-fold faster than those of MASP-2. Moreover, both activated and proenzyme MASP-1 can effectively cleave proenzyme MASP-2. MASP-3, which does not autoactivate, is also cleaved by MASP-1 quite efficiently. The structure of the catalytic region of proenzyme MASP-1 R448Q was solved at 2.5 Å. Proenzyme MASP-1 R448Q readily cleaves synthetic substrates, and it is inhibited by a specific canonical inhibitor developed against active MASP-1, indicating that zymogen MASP-1 fluctuates between an inactive and an active-like conformation. The determined structure provides a feasible explanation for this phenomenon. In summary, autoactivation of MASP-1 is crucial for the activation of MBL/ficolin·MASP complexes, and in the proenzymic phase zymogen MASP-1 controls the process.

Double heterozygous mutations Gln100Leu and His348Gln of the F7 gene in a patient with factor VII deficiency

A 25-year-old Chinese woman who had a history of easy bruising was admitted to hospital due to uncontrolled epistaxis. She showed factor VII activity level of 2% and factor VII antigen level of 4% of the normal value. We detected a novel missense mutation g.8355 A>T (p.Gln100Leu) in the second epidermal growth factor-like (EGF) domain and a g.11482 T>G (p.His348Gln) in the catalytic domain. Although the Gln100 residue is close to the junction of EGF-2 domain with the serine protease domain, we infer that the substitution of polar negatively charged Gln residue at the position 100 with introduction of nonpolar Leu residue may be likely to perturb proper folding, resulting in decreasing factor VII activity.

Molecular dynamics study of hell’s gate globin I (HGbI) from a methanotrophic extremophile: oxygen migration through a large cavity

Hell's gate globin I (HGbI), a heme-containing protein from the extremophile Methylacidiphilum infernorum, has fast oxygen-binding/slow release characteristics due to its distal residues Gln and Tyr. The combination of Gln/Tyr distal iron coordination, adaptation to extreme environmental conditions, and lack of a D helix suggests that ligand migration in HGbI differs from other previously studied globins. Locally enhanced molecular dynamics trajectories of oxygen migration indicate a large internal cavity. This may increase the tendency of oxygen to exit from portals other than the most direct exit from the space near the heme. Oxygen may reside transiently in shallow surface depressions around the exits. Such surface trapping may enhance both oxygen uptake by increasing contact time between molecules, and decrease release by increasing the probability of oxygen reentry from the vicinity of the portal.

Switching the Clientele

The serotonin transporter (SERT) maintains serotonergic neurotransmission via rapid reuptake of serotonin from the synaptic cleft. SERT relies exclusively on the coat protein complex II component SEC24C for endoplasmic reticulum (ER) export. The closely related transporters for noradrenaline and dopamine depend on SEC24D. Here, we show that discrimination between SEC24C and SEC24D is specified by the residue at position +2 downstream from the ER export motif ((607)RI(608) in SERT). Substituting Lys(610) with tyrosine, the corresponding residue found in the noradrenaline and dopamine transporters, switched the SEC24 isoform preference: SERT-K610Y relied solely on SEC24D to reach the cell surface. This analysis was extended to other SLC6 (solute carrier 6) transporter family members: siRNA-dependent depletion of SEC24C, but not of SEC24D, reduced surface levels of the glycine transporter-1a, the betaine/GABA transporter and the GABA transporter-4. Experiments with dominant negative versions of SEC24C and SEC24D recapitulated these findings. We also verified that the presence of two ER export motifs (in concatemers of SERT and GABA transporter-1) supported recruitment of both SEC24C and SEC24D. To the best of our knowledge, this is the first report to document a change in SEC24 specificity by mutation of a single residue in the client protein. Our observations allowed for deducing a rule for SLC6 family members: a hydrophobic residue (Tyr or Val) in the +2 position specifies interaction with SEC24D, and a hydrophilic residue (Lys, Asn, or Gln) recruits SEC24C. Variations in SEC24C are linked to neuropsychiatric disorders. The present findings provide a mechanistic explanation. Variations in SEC24C may translate into distinct surface levels of neurotransmitter transporters.

An engineered fibrinogen variant AαQ328,366P does not polymerise normally, but retains the ability to form α cross-links

A fibrin clot is stabilised through the formation of factor XIIIa-catalysed intermolecular ε-lysyl-γ-glutamyl covalent cross-links between α chains to form α polymers and between γ chains to form γ dimers. In a previous study we characterised fibrinogen Seoul II, a heterozygous dysfibrinogen in which a cross-linking acceptor site in Aα chain, Gln328, was replaced with Pro (AαQ328P). Following on the previous study, we investigated whether the alteration of Gln residues Aα328 and Aα366 affects fibrin polymerisation and α chain cross-linking. We have expressed three recombinant fibrinogens: AαQ328P, AαQ366P, and AαQ328,366P in Chinese hamster ovary cells, purified these fibrinogens from the culture media and performed biochemical tests to see how the introduced changes affect fibrin polymerisation and α chain cross-linking. Thrombin-catalysed fibrin polymerisation of all variants was impaired with the double mutation being the most impaired. In contrast, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis showed α polymer formation with all three engineered proteins. This study demonstrates that AαQ328 and AαQ366 are important for normal fibrin clot formation and in the absence of residues AαQ328 and AαQ366, other Gln residues in the α chain can support FXIIIa-catalysed fibrin cross-linking.