Xiaoli Shi

Structural mechanism of ring-opening reaction of glucose by human serum albumin

Background: Glucose can glycate human serum albumin (HSA), but the mechanism is unknown. Results: Crystal structures of rHSA in the presence of glucose show that glucose is linearized and covalently linked to rHSA. Conclusion: The residues Lys-195 and Lys-199 of rHSA are involved in glucose ring opening. Significance: This work provides a structural mechanism of protein glycation. Glucose reacts with proteins nonenzymatically under physiological conditions. Such glycation is exacerbated in diabetic patients with high levels of blood sugar and induces various complications. Human albumin serum (HSA) is the most abundant protein in plasma and is glycated by glucose. The glycation sites on HSA remain controversial among different studies. Here, we report two protein crystal structures of HSA in complex with either glucose or fructose. These crystal structures reveal the presence of linear forms of sugar for both monosaccharides. The linear form of glucose forms a covalent bond to Lys-195 of HSA, but this is not the case for fructose. Based on these structures, we propose a mechanism for glucose ring opening involving both residues Lys-195 and Lys-199. These results provide mechanistic insights to understand the glucose ring-opening reaction and the glycation of proteins by monosaccharides.

Structural Evidence of Perfluorooctane Sulfonate Transport by Human Serum Albumin

Perfluorooctane sulfonate (PFOS) is a man-made fluorosurfactant and globally persistent organic pollutant. PFOS is mainly distributed in blood with a long half-life for elimination. PFOS was found mainly bound to human serum albumin (HSA) in plasma, the most abundant protein in human blood plasma, which transports a variety of endogenous and exogenous ligands. However, the structural basis of such binding remains unclear. Here, we report the crystal structure of the HSA-PFOS complex and show that PFOS binds to HSA at a molar ratio of 2:1. In addition, PFOS binding renders the HSA structure more compact. Our results provide a structural mechanism to understand the retention of surfactants in human serum.

Structural basis of transport of lysophospholipids by human serum albumin

Lysophospholipids play important roles in cellular signal transduction and are implicated in many biological processes, including tumorigenesis, angiogenesis, immunity, atherosclerosis, arteriosclerosis, cancer and neuronal survival. The intracellular transport of lysophospholipids is through FA (fatty acid)-binding protein. Lysophospholipids are also found in the extracellular space. However, the transport mechanism of lysophospholipids in the extracellular space is unknown. HSA (human serum albumin) is the most abundant carrier protein in blood plasma and plays an important role in determining the absorption, distribution, metabolism and excretion of drugs. In the present study, LPE (lysophosphatidylethanolamine) was used as the ligand to analyse the interaction of lysophospholipids with HSA by fluorescence quenching and crystallography. Fluorescence measurement showed that LPE binds to HSA with a Kd (dissociation constant) of 5.6 microM. The presence of FA (myristate) decreases this binding affinity (Kd of 12.9 microM). Moreover, we determined the crystal structure of HSA in complex with both myristate and LPE and showed that LPE binds at Sudlow site I located in subdomain IIA. LPE occupies two of the three subsites in Sudlow site I, with the LPE acyl chain occupying the hydrophobic bottom of Sudlow site I and the polar head group located at Sudlow site I entrance region pointing to the solvent. This orientation of LPE in HSA suggests that HSA is capable of accommodating other lysophospholipids and phospholipids. The study provides structural information on HSA-lysophospholipid interaction and may facilitate our understanding of the transport and distribution of lysophospholipids.

Structural Insight into Inactivation of Plasminogen Activator Inhibitor-1 by a Small-Molecule Antagonist

Plasminogen activator inhibitor-1 (PAI-1), a serpin, is the physiological inhibitor of tissue-type and urokinase-type plasminogen activators and thus also an inhibitor of fibrinolysis and tissue remodeling. It is a potential therapeutic target in many pathological conditions, including thrombosis and cancer. Several types of PAI-1 antagonist have been developed, but the structural basis for their action has remained largely unknown. Here we report X-ray crystal structure analysis of PAI-1 in complex with a small-molecule antagonist, embelin. We propose a mechanism for embelin-induced rapid conversion of PAI-1 into a substrate for its target proteases and the subsequent slow conversion of PAI-1 into an irreversibly inactivated form. Our work provides structural clues to an understanding of PAI-1 inactivation by small-molecule antagonists and an important step toward the design of drugs targeting PAI-1.

A fluorescent fatty acid probe, DAUDA, selectively displaces two myristates bound in human serum albumin.

11‐(Dansylamino) undecanoic acid (DAUDA) is a dansyl‐type fluorophore and has widely used as a probe to determine the binding site for human serum albumin (HSA). Here, we reported that structure of HSA‐Myristate‐DAUDA ternary complex and identified clearly the presence of two DAUDA molecules at fatty acid (FA) binding site 6 and 7 of HSA, thus showing these two sites are weak FA binding sites. This result also show that DAUDA is an appropriate probe for FA site 6 and 7 on HSA as previous studied, but not a good probe of FA binding site 1 that is likely bilirubin binding site on HSA.

Crystal Structure of the Michaelis Complex between Tissue-type Plasminogen Activator and Plasminogen Activators Inhibitor-1

Background: Recombinant tissue-type plasminogen activator (tPA) is a potent fibrinolytic agent used in clinics and is inactivated by endogenous PAI-1. Results: The crystal structure of the tPA·PAI-1 Michaelis complex was determined. Conclusion: Differences of inhibition of tPA and uPA by PAI-1 are revealed. Significance: This study offers important clues to design a newer generation of tPA thrombolytics with reduced PAI-1 inactivation. Thrombosis is a leading cause of death worldwide. Recombinant tissue-type plasminogen activator (tPA) is the Food and Drug Administration-approved thrombolytic drug. tPA is rapidly inactivated by endogenous plasminogen activator inhibitor-1 (PAI-1). Engineering on tPA to reduce its inhibition by PAI-1 without compromising its thrombolytic effect is a continuous effort. Precise details, with atomic resolution, of the molecular interactions between tPA and PAI-1 remain unknown despite previous extensive studies. Here, we report the crystal structure of the tPA·PAI-1 Michaelis complex, which shows significant differences from the structure of its urokinase-type plasminogen activator analogue, the uPA·PAI-1 Michaelis complex. The PAI-1 reactive center loop adopts a unique kinked conformation. The structure provides detailed interactions between tPA 37- and 60-loops with PAI-1. On the tPA side, the S2 and S1β pockets open up to accommodate PAI-1. This study provides structural basis to understand the specificity of PAI-1 and to design newer generation of thrombolytic agents with reduced PAI-1 inactivation.

Structural Basis for the Interaction between the Golgi Reassembly-stacking Protein GRASP65 and the Golgi Matrix Protein GM130

Background: GRASP65 and GM130 play a key role in Golgi stacking. Results: The crystal structure of the GRASP65-GM130 complex reveals an unexpected binding mode. Conclusion: Both PDZ domains of GRASP65 interact with GM130. A novel mechanism for Golgi stacking is proposed. Significance: A novel interaction mode permits us to propose a new molecular basis for Golgi structure. GM130 and GRASP65 are Golgi peripheral membrane proteins that play a key role in Golgi stacking and vesicle tethering. However, the molecular details of their interaction and their structural role as a functional unit remain unclear. Here, we present the crystal structure of the PDZ domains of GRASP65 in complex with the GM130 C-terminal peptide at 1.96-Å resolution. In contrast to previous findings proposing that GM130 interacts with GRASP65 at the PDZ2 domain only, our crystal structure of the complex indicates that GM130 binds to GRASP65 at two distinct sites concurrently and that both the PDZ1 and PDZ2 domains of GRASP65 participate in this molecular interaction. Mutagenesis experiments support these structural observations and demonstrate that they are required for GRASP65-GM130 association.

Identification and biophysical assessment of the molecular recognition mechanisms between the human haemopoietic cell kinase Src homology domain 3 and ALG-2-interacting protein X

SFKs (Src family kinases) are central regulators of many signalling pathways. Their functions are tightly regulated through SH (Src homology) domain-mediated protein-protein interactions. A yeast two-hybrid screen using SH3 domains as bait identified Alix [ALG-2 (apoptosis-linked gene 2)-interacting protein X] as a novel Hck (haemopoietic cell kinase) SH3 domain interactor. The Alix-Hck-SH3 interaction was confirmed in vitro by a GST (glutathione transferase) pull-down assay and in intact cells by a mammalian two-hybrid assay. Furthermore, the interaction was demonstrated to be biologically relevant in cells. Through biophysical experiments, we then identified the PRR (proline-rich region) motif of Alix that binds Hck-SH3 and determined a dissociation constant of 34.5 μM. Heteronuclear NMR spectroscopy experiments were used to map the Hck-SH3 residues that interact with an ALIX construct containing the V and PRR domains or with the minimum identified interacting motif. Finally, SAXS (small-angle X-ray scattering) analysis showed that the N-terminal PRR of Alix is unfolded, at least before Hck-SH3 recognition. These results indicate that residues outside the canonical PxxP motif of Alix enhance its affinity and selectivity towards Hck-SH3. The structural framework of the Hck-Alix interaction will help to clarify how Hck and Alix assist during virus budding and cell-surface receptor regulation.

Structural recognition mechanisms between human Src homology domain 3 (SH3) and ALG-2-interacting protein X (Alix)

FynR96I physically interacts with NEF by two hybrid ( View interaction )

A specific protein disorder catalyzer of HIV-1 Nef.

The HIV-1 auxiliary protein Nef is required for the onset and progression of AIDS in HIV-1-infected persons. Here, we have deciphered the mode of action of a second-generation inhibitor of Nef, DLC27-14, presenting a competitive IC(50) of ∼16 μM measured by MALDI-TOF experiments. Thermal protein denaturation experiments revealed a negative effect on stability of Nef in the presence of a saturating concentration of the inhibitor. The destabilizing action of DLC27-14 was confirmed by a HIV protease-based experiment, in which the protease sensitivity of DLC27-14-bound Nef was three times as high as that of apo Nef. The only compatible docking modes of action for DLC27-14 suggest that DLC27-14 promotes an opening of two α-helices that would destabilize the Nef core domain. DLC27-14 thus acts as a specific protein disorder catalyzer that destabilizes the folded conformation of the protein. Our results open novel avenues toward the development of next-generation Nef inhibitors.