Simon Terzyan

Structure of the APPL1 BAR-PH domain and characterization of its interaction with Rab5.

APPL1 is an effector of the small GTPase Rab5. Together, they mediate a signal transduction pathway initiated by ligand binding to cell surface receptors. Interaction with Rab5 is confined to the amino (N)‐terminal region of APPL1. We report the crystal structures of human APPL1 N‐terminal BAR‐PH domain motif. The BAR and PH domains, together with a novel linker helix, form an integrated, crescent‐shaped, symmetrical dimer. This BAR–PH interaction is likely conserved in the class of BAR‐PH containing proteins. Biochemical analyses indicate two independent Rab‐binding sites located at the opposite ends of the dimer, where the PH domain directly interacts with Rab5 and Rab21. Besides structurally supporting the PH domain, the BAR domain also contributes to Rab binding through a small surface region in the vicinity of the PH domain. In stark contrast to the helix‐dominated, Rab‐binding domains previously reported, APPL1 PH domain employs β‐strands to interact with Rab5. On the Rab5 side, both switch regions are involved in the interaction. Thus we identified a new binding mode between PH domains and small GTPases.

Structural basis of Rab5-Rabaptin5 interaction in endocytosis

Rab5 is a small GTPase that regulates early endosome fusion. We present here the crystal structure of the Rab5 GTPase domain in complex with a GTP analog and the C-terminal domain of effector Rabaptin5. The proteins form a dyad-symmetric Rab5–Rabaptin52–Rab5 ternary complex with a parallel coiled-coil Rabaptin5 homodimer in the middle. Two Rab5 molecules bind independently to the Rabaptin5 dimer using their switch and interswitch regions. The binding does not involve the Rab complementarity-determining regions. We also present the crystal structures of two distinct forms of GDP–Rab5 complexes, both of which are incompatible with Rabaptin5 binding. One has a dislocated and disordered switch I but a virtually intact switch II, whereas the other has its β-sheet and both switch regions reorganized. Biochemical and functional analyses show that the crystallographically observed Rab5–Rabaptin5 complex also exists in solution, and disruption of this complex by mutation abrogates endosome fusion.

Crystal structure of a glycosylated Fab from an IgM cryoglobulin with properties of a natural proteolytic antibody.

The 2.6 A (1 A=0.1 nm) resolution structure has been determined for the glycosylated Fab (fragment antigen binding) of an IgM (Yvo) obtained from a subject with Waldenströms macroglobulinaemia. Dynamic light scattering was used to estimate the gel point and monitor the formation of an ordered hydroscopic gel of Yvo IgM upon cooling. If a cryoglobulin forms gels in peripheral tissues and organs, the associated swelling and damage to microvasculature can result in considerable morbidity and mortality. The three-dimensional structure of the branched N-linked oligosaccharide associated with the CH1 domain (first constant domain of heavy chain) is reported. The carbohydrate may act to shield part of the lateral surface of the CH1 domain and crowd the junction between the CH1 and CH2 domains, thereby limiting the segmental flexibility of the Fab arms in intact Yvo IgM, especially at low temperatures. Recently, Yvo IgM was shown to have the properties of a naturally occurring proteolytic antibody [Paul, Karle, Planque, Taguchi, Salas, Nishiyama, Handy, Hunter, Edmundson and Hanson (2004) J. Biol. Chem. 279, 39611-39619; Planque, Bangale, Song, Karle, Taguchi, Poindexter, Bick, Edmundson, Nishiyama and Paul (2004) J. Biol Chem. 279, 14024-14032]. The Yvo protein displayed the ability to cleave, by a nucleophilic mechanism, the amide bonds of a variety of serine protease substrates and the gp120 coat protein of HIV. An atypical serine, arginine and glutamate motif is located in the middle of the Yvo antigen-binding site and displays an overall geometry that mimics the classical serine, histidine and aspartate catalytic triad of serine proteases. Our present findings indicate that pre-existing or natural antibodies can utilize at least one novel strategy for the cleavage of peptide bonds.

Inhibition of human γ-glutamyl transpeptidase: development of more potent, physiologically relevant, uncompetitive inhibitors

GGT (γ-glutamyl transpeptidase) is an essential enzyme for maintaining cysteine homoeostasis, leukotriene synthesis, metabolism of glutathione conjugates and catabolism of extracellular glutathione. Overexpression of GGT has been implicated in many pathologies, and clinical inhibitors of GGT are under development for use in the treatment of asthma, cancer and other diseases. Inhibitors are generally characterized using synthetic GGT substrates. The present study of uncompetitive inhibitors of GGT, has revealed that the potency with which compounds inhibit GGT activity in the standard biochemical assay does not correlate with the potency with which they inhibit the physiological reaction catalysed by GGT. Kinetic studies provided insight into the mechanism of inhibition. Modifications to the sulfobenzene or distal benzene ring of the uncompetitive inhibitor OU749 affected activity. One of the most potent inhibitors was identified among a novel group of analogues with an amine group para on the benzosulfonamide ring. New more potent uncompetitive inhibitors of the physiological GGT reaction were found to be less toxic than the glutamine analogues that have been tested clinically. Development of non-toxic inhibitors is essential for exploiting GGT as a therapeutic target.

Crystal structure of GGA2 VHS domain and its implication in plasticity in the ligand binding pocket

Golgi‐localized, γ‐ear‐containing, ARF binding (GGA) proteins regulate intracellular vesicle transport by recognizing sorting signals on the cargo surface in the initial step of the budding process. The VHS (VPS27, Hrs, and STAM) domain of GGA binds with the signal peptides. Here, a crystal structure of the VHS domain of GGA2 is reported at 2.2 Å resolution, which permits a direct comparison with that of homologous proteins, GGA1 and GGA3. Significant structural difference is present in the loop between helices 6 and 7, which forms part of the ligand binding pocket. Intrinsic fluorescence spectroscopic study indicates that this loop undergoes a conformational change upon ligand binding. Thus, the current structure suggests that a conformational change induced by ligand binding occurs in this part of the ligand pocket.

Human γ-Glutamyl Transpeptidase 1 STRUCTURES OF THE FREE ENZYME, INHIBITOR-BOUND TETRAHEDRAL TRANSITION STATES, AND GLUTAMATE-BOUND ENZYME REVEAL NOVEL MOVEMENT WITHIN THE ACTIVE SITE DURING CATALYSIS

Background: γ-Glutamyl transpeptidase 1 (GGT1) plays a role in asthma, reperfusion injury, and cancer. Results: We report four new crystal structures of human GGT1, including the free enzyme, inhibitor-bound transition states, and glutamate-bound enzyme. Conclusion: Novel enzyme-substrate interactions and movement of the catalytic nucleophile, oxyanion hole, and lid loop were revealed. Significance: This structural information is critical for developing GGT1 inhibitors. γ-Glutamyl transpeptidase 1 (GGT1) is a cell surface, N-terminal nucleophile hydrolase that cleaves glutathione and other γ-glutamyl compounds. GGT1 expression is essential in cysteine homeostasis, and its induction has been implicated in the pathology of asthma, reperfusion injury, and cancer. In this study, we report four new crystal structures of human GGT1 (hGGT1) that show conformational changes within the active site as the enzyme progresses from the free enzyme to inhibitor-bound tetrahedral transition states and finally to the glutamate-bound structure prior to the release of this final product of the reaction. The structure of the apoenzyme shows flexibility within the active site. The serine-borate-bound hGGT1 crystal structure demonstrates that serine-borate occupies the active site of the enzyme, resulting in an enzyme-inhibitor complex that replicates the enzymes tetrahedral intermediate/transition state. The structure of GGsTop-bound hGGT1 reveals its interactions with the enzyme and why neutral phosphonate diesters are more potent inhibitors than monoanionic phosphonates. These structures are the first structures for any eukaryotic GGT that include a molecule in the active site covalently bound to the catalytic Thr-381. The glutamate-bound structure shows the conformation of the enzyme prior to release of the final product and reveals novel information regarding the displacement of the main chain atoms that form the oxyanion hole and movement of the lid loop region when the active site is occupied. These data provide new insights into the mechanism of hGGT1-catalyzed reactions and will be invaluable in the development of new classes of hGGT1 inhibitors for therapeutic use.

Comparison of cytochromes b5 from insects and vertebrates.

We report a 1.55 Å X‐ray crystal structure of the heme‐binding domain of cytochrome b5 from Musca domestica (house fly; HF b5), and compare it with previously published structures of the heme‐binding domains of bovine microsomal cytochrome b5 (bMc b5) and rat outer mitochondrial membrane cytochrome b5 (rOM b5). The structural comparison was done in the context of amino acid sequences of all known homologues of the proteins under study. We show that insect b5s contain an extended hydrophobic patch at the base of the heme binding pocket, similar to the one previously shown to stabilize mammalian OM b5s relative to their Mc counterparts. The hydrophobic patch in insects includes a residue with a bulky hydrophobic side chain at position 71 (Met). Replacing Met71 in HF b5 with Ser, the corresponding residue in all known mammalian Mc b5s, is found to substantially destabilize the holoprotein. The destabilization is a consequence of two related factors: (1) a large decrease in apoprotein stability and (2) extension of conformational disruption in the apoprotein beyond the empty heme binding pocket (core 1) and into the heme‐independent folding core (core 2). Analogous changes have previously been shown to accompany replacement of Leu71 in rOM b5 with Ser. That the stabilizing role of Met71 in HF b5 is manifested primarily in the apo state is highlighted by the fact that its crystallographic Cα B factor is modestly larger than that of Ser71 in bMc b5, indicating that it slightly destabilizes local polypeptide conformation when heme is in its binding pocket. Finally, we show that the final unit of secondary structure in the cytochrome b5 heme‐binding domain, a 310 helix known as α6, differs substantially in length and packing interactions not only for different protein isoforms but also for given isoforms from different species. Proteins 2007.

Refinement of the structure of human Rab5a GTPase domain at 1.05 Å resolution

Rab5 is a GTPase that regulates early endosome fusion. Its GTPase domain crystal structure is reported here at 1.05 A resolution in complex with a GTP-analog molecule. It provides the highest resolution three-dimensional model so far obtained for proteins from the Ras-like GTPase family. This study allows extension of structural examination of the GTPase machinery as well as of high-resolution protein structures in general. For example, a buried water-molecule network was observed underneath the switch regions, which is consistent with the functional roles of these regions in the molecular-switching process. Furthermore, residues of multiple conformation and clustered distribution of anisotropic thermal motions of the protein molecule may have general implications for the function of Ras-like GTPases.

Accommodating a nonconservative internal mutation by water-mediated hydrogen bonding between β-sheet strands: a comparison of human and rat type B (mitochondrial) cytochrome b5.

Mammalian type B (mitochondrial) b(5) cytochromes exhibit greater amino acid sequence diversity than their type A (microsomal) counterparts, as exemplified by the type B proteins from human (hCYB5B) and rat (rCYB5B). The comparison of X-ray crystal structures of hCYB5B and rCYB5B reported herein reveals a striking difference in packing involving the five-strand β-sheet, which can be attributed to fully buried residue 21 in strand β4. The greater bulk of Leu21 in hCYB5B in comparison to that of Thr21 in rCYB5B results in a substantial displacement of the first two residues in β5, and consequent loss of two of the three hydrogen bonds between β5 and β4. Hydrogen bonding between the residues is instead mediated by two well-ordered, fully buried water molecules. In a 10 ns molecular dynamics simulation, one of the buried water molecules in the hCYB5B structure exchanged readily with solvent via intermediates having three water molecules sandwiched between β4 and β5. When the buried water molecules were removed prior to a second 10 ns simulation, β4 and β5 formed persistent hydrogen bonds identical to those in rCYB5B, but the Leu21 side chain was forced to adopt a rarely observed conformation. Despite the apparently greater ease of access of water to the interior of hCYB5B than of rCYB5B suggested by these observations, the two proteins exhibit virtually identical stability, dynamic, and redox properties. The results provide new insight into the factors stabilizing the cytochrome b(5) fold.

Characterization of Lys-698 to met substitution in human plasminogen catalytic domain

Streptokinase (SK) is a human plasminogen (Pg) activator secreted by streptococci. The activation mechanism of SK differs from that of physiological Pg activators in that SK is not a protease and cannot proteolytically activate Pg. Instead, it forms a tight complex with Pg that proteolytically activates other Pg molecules. The residue Lys‐698 of human Pg was hypothesized to participate in triggering activation in the SK–Pg complex. Here, we report a study of the Lys‐698 to Met substitution in the catalytic domain of Pg (μPg) containing the proteolytic activation‐resistant background (R561A). While it remains competent in forming a complex with SK, maintaining a comparable equilibration dissociation constant (KD), the recombinant protein shows a nearly 60‐fold reduction in amidolytic activity relative to its R561A background when mixed with native SK. A 2.3 Å crystal structure of this mutant μPg confirmed the correct folding of this recombinant protein. Combined with other biochemical data, these results support the premise that Lys‐698 of human Pg plays a functional role in the so‐called N‐terminal insertion activation mechanism by SK. Proteins 2004.