Guangyu Zhu

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.

Structure of human lanthionine synthetase C-like protein 1 and its interaction with Eps8 and glutathione.

Eukaryotic lanthionine synthetase C-like protein 1 (LanCL1) is homologous to prokaryotic lanthionine cyclases, yet its biochemical functions remain elusive. We report the crystal structures of human LanCL1, both free of and complexed with glutathione, revealing glutathione binding to a zinc ion at the putative active site formed by conserved GxxG motifs. We also demonstrate by in vitro affinity analysis that LanCL1 binds specifically to the SH3 domain of a signaling protein, Eps8. Importantly, expression of LanCL1 mutants defective in Eps8 interaction inhibits nerve growth factor (NGF)-induced neurite outgrowth, providing evidence for the biological significance of this novel interaction in cellular signaling and differentiation.

Crystal structure and mutagenic analysis of GDOsp, a gentisate 1,2-dioxygenase from Silicibacter pomeroyi.

Dioxygenases catalyze dioxygen incorporation into various organic compounds and play a key role in the complex degradation pathway of mono‐ and polycyclic aromatic and hetero‐aromatic compounds. Here we report the crystal structure of gentisate 1,2‐dioxygenase from Silicibacter pomeroyi (GDOsp) at a 2.8 Å resolution. The enzyme possessed a conserved three‐dimensional structure of the bicupin family, forming a homotetramerization. However, each subunit of GDOsp unusually contained two ferrous centers that were located in its two homologous cupin domains, respectively. Further mutagenic analysis indicated that the enzyme activity of GDOsp depends on the microenvironment in both metal‐binding sites. Moreover, homologous structural comparison and functional study on GDOsp variants unveiled a group of functionally essential residues and suggested that the active site of the enzyme is located in the amino‐terminal domain, but could be influenced by changes in the carboxyl domain. Therefore, GDOsp may provide a working model for studying long‐distance communication within a protein (or its complex).

Crystal structure of a carbonyl reductase from Candida parapsilosis with anti-Prelog stereospecificity

A novel short‐chain (S)‐1‐phenyl‐1,2‐ethanediol dehydrogenase (SCR) from Candida parapsilosis exhibits coenzyme specificity for NADPH over NADH. It catalyzes an anti‐Prelog type reaction to reduce 2‐hydroxyacetophenone into (S)‐1‐phenyl‐1,2‐ethanediol. The coding gene was overexpressed in Escherichia coli and the purified protein was crystallized. The crystal structure of the apo‐form was solved to 2.7 Å resolution. This protein forms a homo‐tetramer with a broken 2‐2‐2 symmetry. The overall fold of each SCR subunit is similar to that of the known structures of other homologous alcohol dehydrogenases, although the latter usually form tetramers with perfect 2‐2‐2 symmetries. Additionally, in the apo‐SCR structure, the entrance of the NADPH pocket is blocked by a surface loop. In order to understand the structure–function relationship of SCR, we carried out a number of mutagenesis–enzymatic analyses based on the new structural information. First, mutations of the putative catalytic Ser‐Tyr‐Lys triad confirmed their functional role. Second, truncation of an N‐terminal 31‐residue peptide indicated its role in oligomerization, but not in catalytic activity. Similarly, a V270D point mutation rendered the SCR as a dimer, rather than a tetramer, without affecting the enzymatic activity. Moreover, the S67D/H68D double‐point mutation inside the coenzyme‐binding pocket resulted in a nearly 10‐fold increase and a 20‐fold decrease in the kcat/KM value when NADH and NADPH were used as cofactors, respectively, with kcat remaining essentially the same. This latter result provides a new example of a protein engineering approach to modify the coenzyme specificity in SCR and short‐chain dehydrogenases/reductases in general.

Crystal structure of human GGA1 GAT domain complexed with the GAT-binding domain of Rabaptin5

GGA proteins coordinate the intracellular trafficking of clathrin‐coated vesicles through their interaction with several other proteins. The GAT domain of GGA proteins interacts with ARF, ubiquitin, and Rabaptin5. The GGA–Rabaptin5 interaction is believed to function in the fusion of trans‐Golgi‐derived vesicles to endosomes. We determined the crystal structure of a human GGA1 GAT domain fragment in complex with the Rabaptin5 GAT‐binding domain. In this structure, the Rabaptin5 domain is a 90‐residue‐long helix. At the N‐terminal end, it forms a parallel coiled‐coil homodimer, which binds one GAT domain of GGA1. In the C‐terminal region, it further assembles into a four‐helix bundle tetramer. The Rabaptin5‐binding motif of the GGA1 GAT domain consists of a three‐helix bundle. Thus, the binding between Rabaptin5 and GGA1 GAT domain is based on a helix bundle–helix bundle interaction. The current structural observation is consistent with previously reported mutagenesis data, and its biological relevance is further confirmed by new mutagenesis studies and affinity analysis. The four‐helix bundle structure of Rabaptin5 suggests a functional role in tethering organelles.

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.

Crystal structure of the hexamer of human heat shock factor binding protein 1

Heat shock response (HSR) is a ubiquitous cellular mechanism that copes with a variety of stresses. This response is mediated by a family of transcriptional activators, heat shock factors (HSFs), which are under tight regulation. HSF binding protein 1 (HSBP1) is a negative regulator of HSR and is reported to bind specifically with the active trimeric form of HSF1, thus inhibiting its activity. HSBP1 contains heptad‐repeats in the primary sequence and was believed to stay in a trimer form in solution. We report the crystal structure of the trimerization domain of the M30I/L55P mutant of human HSBP1 at 1.8 Å resolution. In this crystal form, the HSBP1 fragment of residues 6–53 forms a continuous, 11‐turn long helix. The helix self‐associates to form a parallel, symmetrical, triple coiled‐coil helix bundle, which further assembles into a dimer of trimers in a head‐to‐head fashion. Solution study confirmed that the wild‐type HSBP1 shares similar biophysical properties with the crystallized variant. Furthermore, we identified Ser31, which buried its polar side chain in the hydrophobic interior of the helix bundle, as a stability weak‐spot. Substitution of this residue with Ile increases the melting temperature by 24°C, implicating that this conserved serine residue is maintained at position 31 for functional purposes. Proteins 2009.

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.

Analysis of the Interaction between GGA1 GAT Domain and Rabaptin‐5

GGAs are a family of adaptor proteins involved in vesicular transport. As an effector of the small GTPase Arf, GGA interacts using its GAT domain with the GTP-bound form of Arf. The GAT domain is also found to interact with ubiquitin and rabaptin-5. Rabaptin-5 is, in turn, an effector of another small GTPase, Rab5, which regulates early endosome fusion. The interaction between GGAs and rabaptin-5 is likely to take place in a pathway between the trans-Golgi network and early endosomes. This chapter describes in vitro biochemical characterization of the interaction between the GGA1 GAT domain and rabaptin-5. Combining with the complex crystal structure, we reveal that the binding mode is helix bundle-to-helix bundle in nature.