Integrative Modelling Coupled with Ion Mobility Mass Spectrometry Reveals Structural Features of the Clamp Loader in Complex with Single-Stranded DNA Binding Protein

DNA polymerase III, a decameric 420-kDa assembly, simultaneously replicates both strands of the chromosome in Escherichia coli. A subassembly of this holoenzyme, the seven-subunit clamp loader complex, is responsible for loading the sliding clamp (β₂) onto DNA. Here, we use structural information derived from ion mobility mass spectrometry (IM-MS) to build three-dimensional models of one form of the full clamp loader complex, γ₃δδ′ψχ (254 kDa). By probing the interaction between the clamp loader and a single-stranded DNA (ssDNA) binding protein (SSB₄) and by identifying two distinct conformational states, with and without ssDNA, we assemble models of ψχ–SSB₄ (108 kDa) and the clamp loader–SSB₄ (340 kDa) consistent with IM data. A significant increase in measured collision cross-section (~ 10%) of the clamp loader–SSB₄ complex upon DNA binding suggests large conformational rearrangements. This DNA bound conformation represents the active state and, along with the presence of ψχ, stabilises the clamp loader–SSB₄ complex. Overall, this study of a large heteromeric complex analysed by IM-MS, coupled with integrative modelling, highlights the potential of such an approach to reveal structural features of previously unknown complexes of high biological importance.     

Structural Characterization of Pandoraea pnomenusa B-356 Biphenyl Dioxygenase Reveals Features of Potent Polychlorinated Biphenyl-Degrading Enzymes

The oxidative degradation of biphenyl and polychlorinated biphenyls (PCBs) is initiated in Pandoraea pnomenusa B-356 by biphenyl dioxygenase (BPDO_B356). BPDO_B356, a heterohexameric (αβ)₃ Rieske oxygenase (RO), catalyzes the insertion of dioxygen with stereo- and regioselectivity at the 2,3-carbons of biphenyl, and can transform a broad spectrum of PCB congeners. Here we present the X-ray crystal structures of BPDO_B356 with and without its substrate biphenyl 1.6-Å resolution for both structures. In both cases, the Fe(II) has five ligands in a square pyramidal configuration: H233 Nε2, H239 Nε2, D386 Oδ1 and Oδ2, and a single water molecule. Analysis of the active sites of BPDO_B356 and related ROs revealed structural features that likely contribute to the superior PCB-degrading ability of certain BPDOs. First, the active site cavity readily accommodates biphenyl with minimal conformational rearrangement. Second, M231 was predicted to sterically interfere with binding of some PCBs, and substitution of this residue yielded variants that transform 2,2′-dichlorobiphenyl more effectively. Third, in addition to the volume and shape of the active site, residues at the active site entrance also apparently influence substrate preference. Finally, comparison of the conformation of the active site entrance loop among ROs provides a basis for a structure-based classification consistent with a phylogeny derived from amino acid sequence alignments.     

Structural Characterization by Cross-linking Reveals the Detailed Architecture of a Coatomer-related Heptameric Module from the Nuclear Pore Complex

Most cellular processes are orchestrated by macromolecular complexes. However, structural elucidation of these endogenous complexes can be challenging because they frequently contain large numbers of proteins, are compositionally and morphologically heterogeneous, can be dynamic, and are often of low abundance in the cell. Here, we present a strategy for the structural characterization of such complexes that has at its center chemical cross-linking with mass spectrometric readout. In this strategy, we isolate the endogenous complexes using a highly optimized sample preparation protocol and generate a comprehensive, high-quality cross-linking dataset using two complementary cross-linking reagents. We then determine the structure of the complex using a refined integrative method that combines the cross-linking data with information generated from other sources, including electron microscopy, X-ray crystallography, and comparative protein structure modeling. We applied this integrative strategy to determine the structure of the native Nup84 complex, a stable hetero-heptameric assembly (∼600 kDa), 16 copies of which form the outer rings of the 50-MDa nuclear pore complex (NPC) in budding yeast. The unprecedented detail of the Nup84 complex structure reveals previously unseen features in its pentameric structural hub and provides information on the conformational flexibility of the assembly. These additional details further support and augment the protocoatomer hypothesis, which proposes an evolutionary relationship between vesicle coating complexes and the NPC, and indicates a conserved mechanism by which the NPC is anchored in the nuclear envelope.Macromolecular complexes are the building blocks that drive virtually all cellular and biological processes. In each eukaryotic cell, there exist many hundreds of these protein complexes (1–3), the majority of which are still poorly understood in terms of their structures, dynamics, and functions. The classical structure determination approaches of nuclear magnetic resonance, X-ray crystallography, and electron microscopy (EM)¹ remain challenged in attempts to determine the high-resolution structures of large, dynamic, and flexible complexes in a living cell (4). Thus, additional robust and rapid methods are needed, ideally working in concert with these classical approaches, to allow the greatest structural and functional detail in characterizations of macromolecular assemblies.Integrative modeling approaches help address this need, providing powerful tools for determining the structures of endogenous protein complexes (5, 6) by relying on the collection of an extensive experimental dataset, preferably coming from diverse sources (both classical and new) and different levels of resolution. These data are translated into spatial restraints that are used to calculate an ensemble of structures by satisfying the restraints, which in turn can be analyzed and assessed to determine precision and estimate accuracy (5, 7). A major advantage of this approach is that it readily integrates structural data from different methods and a wide range of resolutions, spanning from a few angstroms to dozens of nanometers. This strategy has been successfully applied to a number of protein complexes (8–16). However, it has proven difficult and time-consuming to generate a sufficient number of accurate spatial restraints to enable high-resolution structural characterization; thus, the determination of spatial restraints currently presents a major bottleneck for widespread application of this integrative approach. An important step forward is therefore the development of technologies for collecting high-resolution and information-rich spatial restraints in a rapid and efficient manner, ideally from endogenous complexes isolated directly from living cells.Chemical cross-linking with mass spectrometric readout (CX-MS) (17, 18) has recently emerged as an enabling approach for obtaining residue-specific restraints on the structures of proteins and protein complexes (19–25). In a CX-MS experiment, the purified protein complex is chemically conjugated by a functional group-specific cross-linker, and this is followed by proteolytic digestion and analysis of the resulting peptide mixture by mass spectrometry (MS). However, because of the complexity of the peptide mixtures and low abundance of most of the informative cross-linked species, comprehensive detection of these cross-linked peptides has proven challenging. This challenge increases substantially in studies of endogenous complexes of modest to low abundance, which encompass the great majority of assemblies in any cell (26, 27). In addition, because most cross-linkers used for CX-MS target primary amines, comprehensive detection of cross-links is further limited by the occurrence of lysine, which constitutes only ∼6% of protein sequences, although these lysine residues are generally present on protein surfaces. The use of cross-linkers with different chemistries and reactive groups, especially toward abundant residues, would increase the cross-linking coverage and could be of great help for downstream structural analysis (28).The nuclear pore complex (NPC) is one of the largest protein assemblies in the cell and is the sole mediator of macromolecular transport between the nucleus and the cytoplasm. The NPC is formed by multiple copies of ∼30 different proteins termed nucleoporins (Nups) that are assembled into discrete subcomplexes (8, 29). These building blocks are arranged into eight symmetrical units called spokes that are radially connected to form several concentric rings. The outer rings of the NPC are mainly formed by the Nup84 complex (a conserved complex, termed the Nup107–Nup160 complex in vertebrates). In budding yeast, the Nup84 complex is an essential, Y-shaped assembly of ∼600 kDa that is formed by seven nucleoporins (Nup133, Nup120, Nup145c, Nup85, Nup84, Seh1, and Sec13 in Saccharomyces cerevisiae) (30). The Nup84 complex has been shown to have a common evolutionary origin with vesicle coating complexes (VCCs), such as COPII, COPI, and clathrin (31, 32), but the evolutionary relationships between these VCCs have not been fully delineated. The Nup84 complex has been extensively characterized; several of its components have been analyzed via X-ray crystallography (33, 34), its overall shape has been defined by means of negative-stain electron microscopy (14, 30, 35, 36), and recently efforts were made to define the protein contacts in the Nup84 complex via CX-MS in humans (35) and a thermophilic fungus (37). Finally, we recently used an integrative modeling approach combining domain mapping, negative-stain electron microscopy (38), and publicly available crystal structures to generate a medium-resolution map of the native Nup84 complex (14). However, despite all these efforts, the fine features of the complex, and in particular the intricate domain orientations and contacts within the complex''s hub, remain poorly described.To address these issues, we present here an optimized CX-MS strategy for robust and in-depth structural characterization of endogenous protein complexes. To test the strategy, we generated a comprehensive high-quality CX-MS dataset on the endogenous Nup84 complex using two complementary cross-linkers, disuccinimidyl suberate (DSS) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). Using the resulting cross-linking restraints together with other sources of information (including electron microscopy, X-ray crystallography, and comparative modeling), we computed a detailed structure of the endogenous Nup84 complex. In addition to providing the overall architecture of the yeast Nup84 complex, the resulting structure reveals the previously unknown architecture of its pentameric structural hub. Our results demonstrate that the present approach provides a robust framework for the standardized generation and use of CX-MS spatial restraints toward the structural characterization of endogenous protein complexes.     

Modeling-Dependent Protein Characterization of the Rice Aldehyde Dehydrogenase (ALDH) Superfamily Reveals Distinct Functional and Structural Features

The completion of the rice genome sequence has made it possible to identify and characterize new genes and to perform comparative genomics studies across taxa. The aldehyde dehydrogenase (ALDH) gene superfamily encoding for NAD(P)⁺-dependent enzymes is found in all major plant and animal taxa. However, the characterization of plant ALDHs has lagged behind their animal- and prokaryotic-ALDH homologs. In plants, ALDHs are involved in abiotic stress tolerance, male sterility restoration, embryo development and seed viability and maturation. However, there is still no structural property-dependent functional characterization of ALDH protein superfamily in plants. In this paper, we identify members of the rice ALDH gene superfamily and use the evolutionary nesting events of retrotransposons and protein-modeling–based structural reconstitution to report the genetic and molecular and structural features of each member of the rice ALDH superfamily in abiotic/biotic stress responses and developmental processes. Our results indicate that rice-ALDHs are the most expanded plant ALDHs ever characterized. This work represents the first report of specific structural features mediating functionality of the whole families of ALDHs in an organism ever characterized.     

Structure of the MORN4/Myo3a Tail Complex Reveals MORN Repeats as Protein Binding Modules

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High Affinity Binders to EphA2 Isolated from Abdurin Scaffold Libraries; Characterization,Binding and Tumor Targeting

Abdurins are a novel antibody-like scaffold derived from the engineering of a single isolated CH2 domain of human IgG. Previous studies established the prolonged serum half-life of Abdurins, the result of a retained FcRn binding motif. Here we present data on the construction of large, diverse, phage-display and cell-free DNA display libraries and the isolation of high affinity binders to the cancer target, membrane-bound ephrin receptor tyrosine kinase class A2 (EphA2). Antigen binding regions were created by designing combinatorial libraries into the structural loops and Abdurins were selected using phage display methods. Initial binders were reformatted into new maturation libraries and low nanomolar binders were isolated using cell-free DNA display, CIS display. Further characterization confirmed binding of the Abdurins to both human and murine EphA2 proteins and exclusively to cell lines that expressed EphA2, followed by rapid internalization. Two different EphA2 binders were labeled with ⁶⁴Cu, using a bifunctional MeCOSar chelator, and administered to mice bearing tumors from transplanted human prostate cancer cells, followed by PET/CT imaging. The anti-EphA2 Abdurins localized in the tumors as early as 4 hours after injection and continued to accumulate up to 48 hours when the imaging was completed. These data demonstrate the ability to isolate high affinity binders from the engineered Abdurin scaffold, which retain a long serum half-life, and specifically target tumors in a xenograft model.     

Structure of an Arrestin2-Clathrin Complex Reveals a Novel Clathrin Binding Domain That Modulates Receptor Trafficking

Non-visual arrestins play a pivotal role as adaptor proteins in regulating the signaling and trafficking of multiple classes of receptors. Although arrestin interaction with clathrin, AP-2, and phosphoinositides contributes to receptor trafficking, little is known about the configuration and dynamics of these interactions. Here, we identify a novel interface between arrestin2 and clathrin through x-ray diffraction analysis. The intrinsically disordered clathrin binding box of arrestin2 interacts with a groove between blades 1 and 2 in the clathrin β-propeller domain, whereas an 8-amino acid splice loop found solely in the long isoform of arrestin2 (arrestin2L) interacts with a binding pocket formed by blades 4 and 5 in clathrin. The apposition of the two binding sites in arrestin2L suggests that they are exclusive and may function in higher order macromolecular structures. Biochemical analysis demonstrates direct binding of clathrin to the splice loop in arrestin2L, whereas functional analysis reveals that both binding domains contribute to the receptor-dependent redistribution of arrestin2L to clathrin-coated pits. Mutagenesis studies reveal that the clathrin binding motif in the splice loop is (L/I)₂GXL. Taken together, these data provide a framework for understanding the dynamic interactions between arrestin2 and clathrin and reveal an essential role for this interaction in arrestin-mediated endocytosis.Many transmembrane signaling systems consist of specific G protein-coupled receptors (GPCRs)³ that transduce a diverse array of extracellular stimuli into intracellular signaling events (1). GPCRs modulate the activity of numerous effector molecules and regulate multiple biological functions including neurotransmission, sensory perception, cardiovascular function, development, and cell growth and differentiation (2). To ensure that extracellular stimuli are translated into intracellular signals of appropriate magnitude and duration, these signaling cascades are tightly regulated. GPCRs are subject to three principle modes of regulation; 1) desensitization, in which a receptor becomes refractory to continued stimuli; 2) endocytosis, where receptors are removed from the cell surface; 3) down-regulation, where total receptor levels are decreased (3, 4). Agonist-dependent regulation is primarily mediated by GPCR kinases that specifically phosphorylate activated GPCRs and initiate the recruitment of arrestins. Arrestins are divided into two major classes, visual and non-visual, based on their localization and function. The non-visual arrestins, arrestin2 and 3 (also termed β-arrestin1 and -2, respectively), are broadly distributed and function in multiple processes including GPCR desensitization, trafficking, and signaling (4–6).Initial structural insight on arrestins was provided by the x-ray crystal structure of bovine arrestin1 (7, 8), whereas the crystal structures of C-terminal-truncated (9) and wild type (10) bovine arrestin2 and salamander arrestin4 (11) have also been solved. In general, arrestins are composed of two major domains made up of β strands and connecting loops that are held together by a polar core region consisting of buried salt bridges. It has been proposed that arrestins adopt an active conformation upon binding to phosphorylated receptors, which disrupts the polar core resulting in the release of the C-terminal tail (12). Disruption of the polar core by point mutation of Arg-169 generates a constitutively active arrestin2, which mimics the active state. This mutated arrestin binds to the β₂-adrenergic receptor (β₂AR) in a phosphorylation-independent manner, induces internalization of a δ-opioid receptor lacking phosphorylation sites (13), and has increased binding to clathrin and AP-2 (14).A role for non-visual arrestins in GPCR endocytosis was first described for the β₂AR (15, 16), although it is now evident that arrestins regulate the trafficking of multiple GPCRs as well as additional classes of receptors (4). An early step in this process involves arrestin binding to an activated phosphorylated receptor that enhances arrestin interaction with the endocytic proteins, clathrin, and AP-2 (16, 17). An additional important step in this process involves arrestin interaction with phosphoinositides such as phosphatidylinositol diphosphate and trisphosphate (18). Although the dynamics of these interactions have not been studied, arrestin2 and -3 have been shown to interact specifically and stoichiometrically with clathrin (16). Furthermore, fluorescence microscopy reveals that activated β₂AR, arrestin2, clathrin, and AP-2 all colocalize upon receptor stimulation (16). The primary clathrin binding determinant in arrestin2, LIELD, spans residues 376–380 and is located in an extended disordered loop that immediately precedes the final C-terminal β-strand (10, 19). This region, the clathrin binding box, is consistent with a consensus motif, LϕXϕ(D/E) (where ϕ is a bulky hydrophobic residue, and X represents any polar amino acid), established in other clathrin-binding proteins including AP-2 (20), AP180 (21), amphiphysin (22), and epsin (23). Importantly, the mutation of this motif in arrestin3 and its deletion in arrestin2 significantly disrupts clathrin binding and receptor endocytosis (14, 19). A mutagenesis study of clathrin localized an arrestin binding site to the N-terminal domain of the clathrin heavy chain, specifically residues Glu-89, Lys-96, and Lys-98 (24). Moreover, a crystal structure of clathrin-(1–363) in complex with an arrestin3 peptide (residues 369–381) supports the mutagenesis data and the predicted location of the arrestin-clathrin interaction site (25).To further elucidate the mechanisms involved in mediating arrestin/clathrin interaction, we have determined the crystal structure of clathrin with the short (arrestin2S) and long (arrestin2L) isoforms of arrestin2, which differ by an 8-amino acid insert between β strands 18 and 19 (26). Our results identify an additional and unique interaction encoded in the arrestin2L isoform that is distinct from the previously well characterized interaction involving the LϕXϕ(D/E) motif. Specifically, we observe that the 8 amino acid splice loop in arrestin2L interacts with a pocket formed by blades 4 and 5 in clathrin. Biochemical and cell biological analysis confirm a role for both binding sites in arrestin2L/clathrin interaction and demonstrate an essential role of these interactions in arrestin-mediated GPCR endocytosis.     

Limited Tryptic Proteolysis of the Benzodiazepine Binding Proteins in Different Species Reveals Structural Homologies

Peptide mapping can be used to elucidate further the structural similarities of the benzodiazepine binding proteins in different vertebrate species. Crude synaptic membrane preparations were photoaffinity-labeled with [3H]flunitrazepam and subsequently degraded with various concentrations of trypsin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by fluorography allowed a comparison of the molecular weights of photolabeled peptides in different species. Tryptic degradation led to a common peptide of 40K in all species investigated, a finding indicating that the benzodiazepine binding proteins are structurally homologous in higher bony fishes and tetrapods.     

Discovery and Characterization of a Novel Cyclic Peptide That Effectively Inhibits Ephrin Binding to the EphA4 Receptor and Displays Anti-Angiogenesis Activity

The EphA4 receptor tyrosine kinase regulates a variety of physiological and pathological processes during neural development and the formation of tumor blood vessels; thus, it represents a new and promising therapeutic target. We used a combination of phage peptide display and computer modeling/docking approaches and discovered a novel cyclic nonapeptide, now designated TYY. This peptide selectively inhibits the binding of the ephrinA5 ligand with EphA4 and significantly blocks angiogenesis in a 3D matrigel culture system. Molecular docking reveals that TYY recognizes the same binding pocket on EphA4 that the natural ephrin ligand binds to and that the Tyr3 and Tyr4 side chains of TYY are both critical for the TYY/EphA4 interaction. The discovery of TYY introduces a valuable probe of EphA4 function and a new lead for EphA4-targeted therapeutic development.     

Crystal Structure of Human Dual-Specificity Tyrosine-Regulated Kinase 3 Reveals New Structural Features and Insights into its Auto-phosphorylation

Dual-specificity tyrosine-regulated kinases (DYRKs) auto-phosphorylate a critical tyrosine residue in their activation loop and phosphorylate their substrate on serine and threonine residues. The auto-phosphorylation occurs intramolecularly and is a one-off event. DYRK3 is selectively expressed at a high level in hematopoietic cells and attenuates erythroblast development, leading to anemia. In the present study, we determined the crystal structure of the mature form of human DYRK3 in complex with harmine, an ATP competitive inhibitor. The crystal structure revealed a phosphorylation site, residue S350, whose phosphorylation increases the stability of DYRK3 and enhances its kinase activity. In addition, our structural and biochemical assays suggest that the N-terminal auto-phosphorylation accessory domain stabilizes the DYRK3 protein, followed by auto-phosphorylation of the tyrosine of the activation loop, which is important for kinase activity. Finally, our docking analysis provides information for the design of novel and potent therapeutics to treat anemia.     

Serendipitous Fatty Acid Binding Reveals the Structural Determinants for Ligand Recognition in Apolipoprotein M

Apolipoprotein M (ApoM) is a 25-kDa HDL-associated apolipoprotein and a member of the lipocalin family of proteins. Mature apoM retains its signal peptide, which serves as a lipid anchor attaching apoM to the lipoproteins, thereby keeping it in the circulation. Studies in mice have suggested apoM to be antiatherogenic, but its physiological function is yet unknown. We have now determined the 1.95 Å resolution crystal structure of recombinant human apoM expressed in Escherichia coli and made the unexpected discovery that apoM, although refolded from inclusion bodies, was in complex with fatty acids containing 14, 16 or 18 carbon atoms. ApoM displays the typical lipocalin fold characterised by an eight-stranded antiparallel β-barrel that encloses an internal ligand-binding pocket. The crystal structures of two different complexes provide a detailed picture of the ligand-binding determinants of apoM. Additional fatty acid- and lipid-binding studies with apoM and the mutants apoM^W47F and apoM^W100F showed that sphingosine-1-phosphate is able to displace the bound fatty acids and efficiently quenched the intrinsic fluorescence with an IC₅₀ of 0.90 μM. Whereas the fatty acids bound in the crystal structure could be a mere consequence of recombinant protein production, the observed binding of sphingosine-1-phosphate might provide a key to a better understanding of the physiological function of apoM.     

Biochemical and Structural Characterization of the Ubiquitin-Conjugating Enzyme UBE2W Reveals the Formation of a Noncovalent Homodimer

The biochemical and structural characterization of ubiquitin-conjugating enzymes (E2s) over the past 30 years has fostered important insights into ubiquitin transfer mechanisms. Although many of these enzymes share high sequence and structural conservation, their functional roles in the cell are decidedly diverse. Here, we report that the mono-ubiquitinating E2 UBE2W forms a homodimer using two distinct protein surfaces. Dimerization is primarily driven by residues in the ß-sheet region and Loops 4 and 7 of the catalytic domain. Mutation of two residues in the catalytic domain of UBE2W is capable of disrupting UBE2W homodimer formation, however, we find that dimerization of this E2 is not required for its ubiquitin transfer activity. In addition, residues in the C-terminal region, although not compulsory for the dimerization of UBE2W, play an ancillary role in the dimer interface. In all current E2 structures, the C-terminal helix of the UBC domain is at least 15Å away from the primary dimerization surface shown here for UBE2W. This leads to the proposal that the C-terminal region of UBE2W adopts a noncanonical position that places it closer to the UBC ß-sheet, providing the first indication that at least some E2s adopt C-terminal conformations different from the canonical structures observed to date.     

Small molecules can selectively inhibit ephrin binding to the EphA4 and EphA2 receptors

The erythropoietin-producing hepatocellular (Eph) family of receptor tyrosine kinases regulates a multitude of physiological and pathological processes. Despite the numerous possible research and therapeutic applications of agents capable of modulating Eph receptor function, no small molecule inhibitors targeting the extracellular domain of these receptors have been identified. We have performed a high throughput screen to search for small molecules that inhibit ligand binding to the extracellular domain of the EphA4 receptor. This yielded a 2,5-dimethylpyrrolyl benzoic acid derivative able to inhibit the interaction of EphA4 with a peptide ligand as well as the natural ephrin ligands. Evaluation of a series of analogs identified an isomer with similar inhibitory properties and other less potent compounds. The two isomeric compounds act as competitive inhibitors, suggesting that they target the high affinity ligand-binding pocket of EphA4 and inhibit ephrin-A5 binding to EphA4 with K(i) values of 7 and 9 mum in enzyme-linked immunosorbent assays. Interestingly, despite the ability of each ephrin ligand to promiscuously bind many Eph receptors, the two compounds selectively target EphA4 and the closely related EphA2 receptor. The compounds also inhibit ephrin-induced phosphorylation of EphA4 and EphA2 in cells, without affecting cell viability or the phosphorylation of other receptor tyrosine kinases. Furthermore, the compounds inhibit EphA4-mediated growth cone collapse in retinal explants and EphA2-dependent retraction of the cell periphery in prostate cancer cells. These data demonstrate that the Eph receptor-ephrin interface can be targeted by inhibitory small molecules and suggest that the two compounds identified will be useful to discriminate the activities of EphA4 and EphA2 from those of other co-expressed Eph receptors that are activated by the same ephrin ligands. Furthermore, the newly identified inhibitors represent possible leads for the development of therapies to treat pathologies in which EphA4 and EphA2 are involved, including nerve injuries and cancer.     

Rapid Binding of Plasminogen to Streptokinase in a Catalytic Complex Reveals a Three-step Mechanism

Rapid kinetics demonstrate a three-step pathway of streptokinase (SK) binding to plasminogen (Pg), the zymogen of plasmin (Pm). Formation of a fluorescently silent encounter complex is followed by two conformational tightening steps reported by fluorescence quenches. Forward reactions were defined by time courses of biphasic quenching during complex formation between SK or its COOH-terminal Lys⁴¹⁴ deletion mutant (SKΔK414) and active site-labeled [Lys]Pg ([5-(acetamido)fluorescein]-d-Phe-Phe-Arg-[Lys]Pg ([5F]FFR-[Lys]Pg)) and by the SK dependences of the quench rates. Active site-blocked Pm rapidly displaced [5F]FFR-[Lys]Pg from the complex. The encounter and final SK·[5F]FFR-[Lys]Pg complexes were weakened similarly by SK Lys⁴¹⁴ deletion and blocking of lysine-binding sites (LBSs) on Pg kringles with 6-aminohexanoic acid or benzamidine. Forward and reverse rates for both tightening steps were unaffected by 6-aminohexanoic acid, whereas benzamidine released constraints on the first conformational tightening. This indicated that binding of SK Lys⁴¹⁴ to Pg kringle 4 plays a role in recognition of Pg by SK. The substantially lower affinity of the final SK·Pg complex compared with SK·Pm is characterized by a ∼25-fold weaker encounter complex and ∼40-fold faster off-rates for the second conformational step. The results suggest that effective Pg encounter requires SK Lys⁴¹⁴ engagement and significant non-LBS interactions with the protease domain, whereas Pm binding additionally requires contributions of other lysines. This difference may be responsible for the lower affinity of the SK·Pg complex and the expression of a weaker “pro”-exosite for binding of a second Pg in the substrate mode compared with SK·Pm.     

Structural Insight into Evolution of the Quinone Binding Site in Complex II

Biochemistry (Moscow) - The Complex II family encompasses membrane bound succinate:quinones reductases and quinol:fumarate reductases that catalyze interconversion of succinate and fumarate coupled...