Identification of a new epitope in uPAR as a target for the cancer therapeutic monoclonal antibody ATN-658, a structural homolog of the uPAR binding integrin CD11b (αM).

The urokinase plasminogen activator receptor (uPAR) plays a role in tumor progression and has been proposed as a target for the treatment of cancer. We recently described the development of a novel humanized monoclonal antibody that targets uPAR and has anti-tumor activity in multiple xenograft animal tumor models. This antibody, ATN-658, does not inhibit ligand binding (i.e. uPA and vitronectin) to uPAR and its mechanism of action remains unclear. As a first step in understanding the anti-tumor activity of ATN-658, we set out to identify the epitope on uPAR to which ATN-658 binds. Guided by comparisons between primate and human uPAR, epitope mapping studies were performed using several orthogonal techniques. Systematic site directed and alanine scanning mutagenesis identified the region of aa 268-275 of uPAR as the epitope for ATN-658. No known function has previously been attributed to this epitope Structural insights into epitope recognition were obtained from structural studies of the Fab fragment of ATN-658 bound to uPAR. The structure shows that the ATN-658 binds to the DIII domain of uPAR, close to the C-terminus of the receptor, corroborating the epitope mapping results. Intriguingly, when bound to uPAR, the complementarity determining region (CDR) regions of ATN-658 closely mimic the binding regions of the integrin CD11b (αM), a previously identified uPAR ligand thought to be involved in leukocyte rolling, migration and complement fixation with no known role in tumor progression of solid tumors. These studies reveal a new functional epitope on uPAR involved in tumor progression and demonstrate a previously unrecognized strategy for the therapeutic targeting of uPAR.

Structure of catalytic domain of Matriptase in complex with Sunflower trypsin inhibitor-1.

BACKGROUND: Matriptase is a type II transmembrane serine protease that is found on the surfaces of epithelial cells and certain cancer cells. Matriptase has been implicated in the degradation of certain extracellular matrix components as well as the activation of various cellular proteins and proteases, including hepatocyte growth factor and urokinase. Sunflower trypsin inhibitor-1 (SFTI-1), a cyclic peptide inhibitor originally isolated from sunflower seeds, exhibits potent inhibitory activity toward matriptase. RESULTS: We have engineered and produced recombinant proteins of the matriptase protease domain, and have determined the crystal structures of the protease:SFTI-1 complex at 2.0 Å as well as the protease:benzamidine complex at 1.2 Å. These structures elaborate the structural basis of substrate selectivity of matriptase, and show that the matriptase S1 substrate specificity pocket is larger enough to allow movement of benzamidine inside the S1 pocket. Our study also reveals that SFTI-1 binds to matriptase in a way similar to its binding to trypsin despite the significantly different isoelectric points of the two proteins (5.6 vs. 8.2). CONCLUSIONS: This work helps to define the structural basis of substrate specificity of matriptase and the interactions between the inhibitor and protease. The complex structure also provides a structural template for designing new SFTI-1 derivatives with better potency and selectivity against matriptase and other proteases.

Trp2313-His2315 of factor VIII C2 domain is involved in membrane binding: structure of a complex between the C2 domain and an inhibitor of membrane binding.

Factor VIII (FVIII) plays a critical role in blood coagulation by forming the tenase complex with factor IXa and calcium ions on a membrane surface containing negatively charged phospholipids. The tenase complex activates factor X during blood coagulation. The carboxyl-terminal C2 domain of FVIII is the main membrane-binding and von Willebrand factor-binding region of the protein. Mutations of FVIII cause hemophilia A, whereas elevation of FVIII activity is a risk factor for thromboembolic diseases. The C2 domain-membrane interaction has been proposed as a target of intervention for regulation of blood coagulation. A number of molecules that interrupt FVIII or factor V (FV) binding to cell membranes have been identified through high throughput screening or structure-based design. We report crystal structures of the FVIII C2 domain under three new crystallization conditions, and a high resolution (1.15 A) crystal structure of the FVIII C2 domain bound to a small molecular inhibitor. The latter structure shows that the inhibitor binds to the surface of an exposed beta-strand of the C2 domain, Trp(2313)-His(2315). This result indicates that the Trp(2313)-His(2315) segment is an important constituent of the membrane-binding motif and provides a model to understand the molecular mechanism of the C2 domain membrane interaction.

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.

Structure of full-length ubiquitin-conjugating enzyme E2-25K (huntingtin-interacting protein 2).

The ubiquitin-conjugating enzyme E2-25K has been identified as a huntingtin (the key protein in Huntington's disease) interacting protein and has been shown to play a role in mediating the toxicity of Abeta, the principal protein involved in Alzheimer's disease pathogenesis. E2-25K is a dual-domain protein with an ubiquitin-associated (UBA) domain as well as a conserved ubiquitin-conjugating (UBC) domain which catalyzes the formation of a covalent bond between the C-terminal glycine of an ubiquitin molecule and the -amine of a lysine residue on the acceptor protein as part of the ubiquitin-proteasome pathway. The crystal structures of E2-25K M172A mutant protein at pH 6.5 and pH 8.5 were determined to 1.9 and 2.2 A resolution, respectively. Examination of the structures revealed domain-domain interactions between the UBC and UBA domains which have not previously been reported.

Structure of an insect epsilon class glutathione S-transferase from the malaria vector Anopheles gambiae provides an explanation for the high DDT-detoxifying activity.

Glutathione S-transferases (GSTs), a major family of detoxifying enzymes, play a pivotal role in insecticide resistance in insects. In the malaria vector Anopheles gambiae, insect-specific epsilon class GSTs are associated with resistance to the organochlorine insecticide DDT [1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane]. Five of the eight class members have elevated expression levels in a DDT resistant strain. agGSTe2 is considered the most important GST in conferring DDT resistance in A. gambiae, and is the only member of the epsilon class with confirmed DDT-metabolizing activity. A delta class GST from the same species shows marginal DDT-metabolizing activity but the activity of agGSTe2 is approximately 350x higher than the delta class agGST1-6. To investigate its catalytic mechanism and the molecular basis of its unusually high DDT-metabolizing ability, three agGSTe2 crystal structures including one apo form and two binary complex forms with the co-factor glutathione (GSH) or the inhibitor S-hexylglutathione (GTX) have been solved with a resolution up to 1.4A. The structure of agGSTe2 shows the canonical GST fold with a highly conserved N-domain and a less conserved C-domain. The binding of GSH or GTX does not induce significant conformational changes in the protein. The modeling of DDT into the putative DDT-binding pocket suggests that DDT is likely to be converted to DDE [1,1-dichloro-2,2-bis-(p-chlorophenyl)ethylene] through an elimination reaction triggered by the nucleophilic attack of the thiolate group of GS(-) on the beta-hydrogen of DDT. The comparison with the less active agGST1-6 provides the structural evidence for its high DDT-detoxifying activity. In short, this is achieved through the inclination of the upper part of H4 helix (H4'' helix), which brings residues Arg112, Glu116, and Phe120 closer to the GSH-binding site resulting in a more efficient GS(-)-stabilizing hydrogen-bond-network and higher DDT-binding affinity.

Atomic resolution structure of cucurmosin, a novel type 1 ribosome-inactivating protein from the sarcocarp of Cucurbita moschata.

A novel type 1 ribosome-inactivating protein (RIP) designated cucurmosin was isolated from the sarcocarp of Cucurbita moschata (pumpkin). Besides rRNA N-glycosidase activity, cucurmosin exhibits strong cytotoxicities to three cancer cell lines of both human and murine origins, but low toxicity to normal cells. Plant genomic DNA extracted from the tender leaves was amplified by PCR between primers based on the N-terminal sequence and X-ray sequence of the C-terminal. The complete mature protein sequence was obtained from N-terminal protein sequencing and partial DNA sequencing, confirmed by high resolution crystal structure analysis. The crystal structure of cucurmosin has been determined at 1.04A, a resolution that has never been achieved before for any RIP. The structure contains two domains: a large N-terminal domain composed of seven alpha-helices and eight beta-strands, and a smaller C-terminal domain consisting of three alpha-helices and two beta-strands. The high resolution structure established a glycosylation pattern of GlcNAc(2)Man(3)Xyl. Asn225 was identified as a glycosylation site. Residues Tyr70, Tyr109, Glu158 and Arg161 define the active site of cucurmosin as an RNA N-glycosidase. The structural basis of cytotoxicity difference between cucurmosin and trichosanthin is discussed.

A new drug binding subsite on human serum albumin and drug-drug interaction studied by X-ray crystallography.

3'-Azido-3'-deoxythymidine (AZT) is the first clinically effective drug for the treatment of human immunodeficiency virus infection. The drug interaction with human serum albumin (HSA) has been an important component in understanding its mechanism of action, especially in drug distribution and in drug-drug interaction on HSA in the case of multi-drug therapy. We present here crystal structures of a ternary HSA-Myr-AZT complex and a quaternary HSA-Myr-AZT-SAL complex (Myr, myristate; SAL, salicylic acid). From this study, a new drug binding subsite on HSA Sudlow site 1 was identified. The presence of fatty acid is needed for the creation of this subsite due to fatty acid induced conformational changes of HSA. Thus, the Sudlow site 1 of HSA can be divided into three non-overlapped subsites: a SAL subsite, an indomethacin subsite and an AZT subsite. Binding of a drug to HSA often influences simultaneous binding of other drugs. From the HSA-Myr-AZT-SAL complex structure, we observed the coexistence of two drugs (AZT and SAL) in Sudlow site 1 and the competition between these two drugs in subdomain IB. These results provide new structural information on HSA-drug interaction and drug-drug interaction on HSA.

1,7-Dihydr-oxy-2,3,4-trimeth-oxy-9H-xanthen-9-one monohydrate from Halenia elliptica.

The title compound, C(16)H(14)O(7)·H(2)O, possesses a planar three-ring skeleton; its carbonyl, one of the two hydroxy and two of the three methoxy O atoms and the water mol-ecule form hydrogen bonds, giving rise to a layer structure.

X-ray sequence and crystal structure of luffaculin 1, a novel type 1 ribosome-inactivating protein.

BACKGROUND: Protein sequence can be obtained through Edman degradation, mass spectrometry, or cDNA sequencing. High resolution X-ray crystallography can also be used to derive protein sequence information, but faces the difficulty in distinguishing the Asp/Asn, Glu/Gln, and Val/Thr pairs. Luffaculin 1 is a new type 1 ribosome-inactivating protein (RIP) isolated from the seeds of Luffa acutangula. Besides rRNA N-glycosidase activity, luffaculin 1 also demonstrates activities including inhibiting tumor cells' proliferation and inducing tumor cells' differentiation. RESULTS: The crystal structure of luffaculin 1 was determined at 1.4 A resolution. Its amino-acid sequence was derived from this high resolution structure using the following criteria: 1) high resolution electron density; 2) comparison of electron density between two molecules that exist in the same crystal; 3) evaluation of the chemical environment of residues to break down the sequence assignment ambiguity in residue pairs Glu/Gln, Asp/Asn, and Val/Thr; 4) comparison with sequences of the homologous proteins. Using the criteria 1 and 2, 66% of the residues can be assigned. By incorporating with criterion 3, 86% of the residues were assigned, suggesting the effectiveness of chemical environment evaluation in breaking down residue ambiguity. In total, 94% of the luffaculin 1 sequence was assigned with high confidence using this improved X-ray sequencing strategy. Two N-acetylglucosamine moieties, linked respectively to the residues Asn77 and Asn84, can be identified in the structure. Residues Tyr70, Tyr110, Glu159 and Arg162 define the active site of luffaculin 1 as an RNA N-glycosidase. CONCLUSION: X-ray sequencing method can be effective to derive sequence information of proteins. The evaluation of the chemical environment of residues is a useful method to break down the assignment ambiguity in Glu/Gln, Asp/Asn, and Val/Thr pairs. The sequence and the crystal structure confirm that luffaculin 1 is a new type 1 RIP.

X-ray crystal structure and antioxidant activity of salidroside, a phenylethanoid glycoside.

The X-ray single-crystal structure of natural salidroside (=2-(4-hydroxyphenyl)ethyl beta-D-glucopyranoside; 1), isolated from Cistanche deserticola, is reported for the first time, as well as its absolute configuration. The radical-scavenging activity of 1 towards the superoxide radical anion (O*2-) was determined experimentally by chemiluminescence measurements of the pyrogallol-luminol system, and compared to that of the corresponding aglycone, i.e., tyrosol (=4-(2-hydroxyethyl)phenol; 2).

An anti-urokinase plasminogen activator receptor (uPAR) antibody: crystal structure and binding epitope.

Human urokinase-type plasminogen activator receptor (uPAR/CD87) is expressed at the invasive interface of the tumor-stromal microenvironment in many human cancers and interacts with a wide array of extracellular molecules. An anti-uPAR antibody (ATN615) was prepared using hybridoma technology. This antibody binds to uPAR in vitro with high affinity (K(d) approximately 1 nM) and does not interfere with uPA binding to uPAR. Here we report the crystal structure of the Fab fragment of ATN615 at 1.77 A and the analysis of ATN615-suPAR-ATF structure that was previously determined, emphasizing the ATN615-suPAR interaction. The complementarity determining regions (CDRs) of ATN615 consist of a high percentage of aromatic residues, and form a relatively flat and undulating surface. The ATN615 Fab fragment recognizes domain 3 of suPAR. The antibody-antigen recognition involves 11 suPAR residues and 12 Fab residues from five CDRs. Structural data suggest that Pro188, Asn190, Gly191, and Arg192 residues of uPAR are the key residues for the antibody recognition, while Pro189 and Arg192 render specificity of ATN615 for human uPAR. Interestingly, this antibody-antigen interface has a small contact area, mainly polar interaction with little hydrophobic character, yet has high binding strength. Furthermore, several solvent molecules (assigned as polyethylene glycols) were clearly visible in the binding interface between antibody and antigen, suggesting that solvent molecules may be important for the maximal binding between suPAR and ATN615 Fab. ATN615 undergoes small but noticeable changes in its CDR region upon antigen binding.

Structure of the SH3 domain of human osteoclast-stimulating factor at atomic resolution.

Osteoclast-stimulating factor (OSF) is an intracellular signaling protein, produced by osteoclasts themselves, that enhances osteoclast formation and bone resorption. It is thought to act via an Src-related signaling pathway and contains SH3 and ankyrin-repeat domains which are involved in protein-protein interactions. As part of a structure-based anti-bone-loss drug-design program, the atomic resolution X-ray structure of the recombinant human OSF SH3 domain (hOSF-SH3) has been determined. The domain, residues 12-72, yielded crystals that diffracted to the ultrahigh resolution of 1.07 A. The overall structure shows a characteristic SH3 fold consisting of two perpendicular beta-sheets that form a beta-barrel. Structure-based sequence alignment reveals that the putative proline-rich peptide-binding site of hOSF-SH3 consists of (i) residues that are highly conserved in the SH3-domain family, including residues Tyr21, Phe23, Trp49, Pro62, Asn64 and Tyr65, and (ii) residues that are less conserved and/or even specific to hOSF, including Thr22, Arg26, Thr27, Glu30, Asp46, Thr47, Asn48 and Leu60, which might be key to designing specific inhibitors for hOSF to fight osteoporosis and related bone-loss diseases. There are a total of 13 well defined water molecules forming hydrogen bonds with the above residues in and around the peptide-binding pocket. Some of those water molecules might be important for drug-design approaches. The hOSF-SH3 structure at atomic resolution provides an accurate framework for structure-based design of its inhibitors.

Mutation of a conserved tryptophan in the chitin-binding cleft of Serratia marcescens chitinase A enhances transglycosylation.

Family 18 chitinases have the signature peptide DGXDXDXE forming the fourth beta-strand in the (beta/alpha)8-barrel of their catalytic domain. The carboxyl-end glutamic acid, E315 in Serratia marcescens chitinase A, serves as the acid/base during chitin hydrolysis, and the side-chain of the preceding aspartic acid, D313, helps to position correctly the N-acetyl moiety of the glycosyl sugar undergoing hydrolysis. Chitin substrates are bound within a long cleft across the top of the barrel, whose floor consists of aromatic residues that hydrophobically stack with every other GlcNAc. Alanine substitution of the conserved Trp167 at the -3 subsite in Serratia marcescens chitinase A enhanced transglycosylation. Higher oligosaccharides were formed from both chitin tetra- and pentasaccharide, and the only hydrolytic product from chitin trisaccharide was the disaccharide. Greater retention of the glycosyl fragment at the active site of the -3 mutant of Serratia marcescens chitinase A might favor transglycosylation due to a stabilized conformation of its D313.

The hyperthermophile protein Sso10a is a dimer of winged helix DNA-binding domains linked by an antiparallel coiled coil rod.

Sso10a is a member of a group of DNA-binding proteins thought to be important in chromatin structure and regulation in the hyperthermophilic archaeon Sulfolobus solfataricus. We have determined the structure of Sso10a to 1.47A resolution directly with unlabelled native crystals by a novel approach using sulfur single-wavelength anomalous scattering (SAS) from a chromium X-ray source. The 95 amino acid residue protein contains a winged helix DNA-binding domain with an extended C-terminal alpha-helix that leads to dimerization by forming a two-stranded, antiparallel coiled-coil rod. The winged helix domains are at opposite ends of the extended coiled coil with two putative DNA-recognition helices separated by 55A and rotated by 83 degrees. Formation of stable dimers in solution is demonstrated by both analytical ultracentrifugation and differential scanning calorimetry. With a T0 of 109 degrees C, Sso10a is one of the most stable two-stranded coiled coils known. The coiled coil contains a rare aspartate residue (D69) in the normally hydrophobic d position of the heptad repeat, with two aspartate-lysine (d-g') interhelical ion pairs in the symmetrical dimer. Mutation of D69 to alanine resulted in an increase in thermal stability, indicating that destabilization resulting from the partially buried aspartate residue cannot be offset by ion pair formation. Possible DNA-binding interactions are discussed on the basis of comparisons to other winged helix proteins. The structure of Sso10a provides insight into the structures of the conserved domain represented by COG3432, a group of more than 20 hypothetical transcriptional regulators coded in the genomic sequences of both crenarchaeota and euryarchaeota.

High resolution crystal structure of human Rab9 GTPase: a novel antiviral drug target.

Rab GTPases and their effectors facilitate vesicular transport by tethering donor vesicles to their respective target membranes. Rab9 mediates late endosome to trans-Golgi transport and has recently been found to be a key cellular component for human immunodeficiency virus-1, Ebola, Marburg, and measles virus replication, suggesting that it may be a novel target in the development of broad spectrum antiviral drugs. As part of our structure-based drug design program, we have determined the crystal structure of a C-terminally truncated human Rab9 (residues 1-177) to 1.25-A resolution. The overall structure shows a characteristic nucleotide binding fold consisting of a six-stranded beta-sheet surrounded by five alpha-helices with a tightly bound GDP molecule in the active site. Structure-based sequence alignment of Rab9 with other Rab proteins reveals that its active site consists of residues highly conserved in the Rab GTPase family, implying a common catalytic mechanism. However, Rab9 contains seven regions that are significantly different in conformation from other Rab proteins. Some of those regions coincide with putative effector-binding sites and switch I and switch II regions identified by structure/sequence alignments. The Rab9 structure at near atomic resolution provides an excellent model for structure-based antiviral drug design.

Crystal structure of type IIE restriction endonuclease EcoRII reveals an autoinhibition mechanism by a novel effector-binding fold.

EcoRII is a type IIE restriction endonuclease that interacts with two copies of the DNA recognition sequence 5'CCWGG, one being the actual target of cleavage, the other serving as the allosteric effector. The mode of enzyme activation by effector binding is unknown. To investigate the molecular basis of activation and cleavage mechanisms by EcoRII, the crystal structure of EcoRII mutant R88A has been solved at 2.1A resolution. The EcoRII monomer has two domains linked through a hinge loop. The N-terminal effector-binding domain has a novel DNA recognition fold with a prominent cleft. The C-terminal catalytic domain has a restriction endonuclease-like fold. Structure-based sequence alignment identified the putative catalytic site of EcoRII that is spatially blocked by the N-terminal domain. The structure together with the earlier characterized EcoRII enzyme activity enhancement in the absence of its N-terminal domain reveal an autoinhibition/activation mechanism of enzyme activity mediated by a novel effector-binding fold. This is the first case of autoinhibition, a mechanism described for many transcription factors and signal transducing proteins, of a restriction endonuclease.

Structure of an insect delta-class glutathione S-transferase from a DDT-resistant strain of the malaria vector Anopheles gambiae.

Glutathione S-transferases (GSTs) are a major family of detoxification enzymes which possess a wide range of substrate specificities. Most organisms possess many GSTs belonging to multiple classes. Interest in GSTs in insects is focused on their role in insecticide resistance; many resistant insects have elevated levels of GST activity. In the malaria vector Anopheles gambiae, elevated GST levels are associated with resistance to the organochlorine insecticide DDT [1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane]. This mosquito is the source of an insect GST, agGSTd1-6, which metabolizes DDT and is inhibited by a number of pyrethroid insecticides. The crystal structure of agGSTd1-6 in complex with its inhibitor S-hexyl glutathione has been determined and refined at 2.0 A resolution. The structure adopts a classical GST fold and is similar to those of other insect delta-class GSTs, implying a common conjugation mechanism. A structure-based model for the binding of DDT to agGSTd1-6 reveals two subpockets in the hydrophobic binding site (H-site), each accommodating one planar p-chlorophenyl ring.

A single mutation of restriction endonuclease EcoRII led to a new crystal form that diffracts to 2.1 A resolution.

R88A, a mutant of the type IIE restriction endonuclease EcoRII, has been crystallized in space group P2(1), with unit-cell parameters a = 58.7, b = 92.4, c = 88.3 A, beta = 108.1 degrees. There are two monomers in the asymmetric unit and the solvent content is estimated to be 50% by volume. The crystals diffract to 2.1 A resolution, which is much higher than that of the wild type, which diffracted to 2.8 A resolution. The mutant crystals have been used in the identification of an excellent heavy-atom derivative.