Amphipathic Motifs Regulate N-BAR Protein Endophilin B1 Auto-inhibition and Drive Membrane Remodeling.

Membrane remodeling is a common theme in a variety of cellular processes. Here, we investigated membrane remodeling N-BAR protein endophilin B1, a critical player in diverse intracellular trafficking events, including mitochondrial and Golgi fission, and apoptosis. We find that endophilin B1 assembles into helical scaffolds on membranes, and that both membrane binding and assembly are driven by interactions between N-terminal helix H0 and the lipid bilayer. Furthermore, we find that endophilin B1 membrane remodeling is auto-inhibited and identify direct SH3 domain-H0 interactions as the underlying mechanism. Our results indicate that lipid composition plays a role in dictating endophilin B1 activity. Taken together, this study provides insight into a poorly understood N-BAR protein family member and highlights molecular mechanisms that may be general for the regulation of membrane remodeling. Our work suggests that interplay between membrane lipids and membrane interacting proteins facilitates spatial and temporal coordination of membrane remodeling.

Structure-guided design of a potent peptide inhibitor targeting the interaction between CRK and ABL kinase.

CT-10 regulator of kinase (CRK) proteins play important roles in human cancer metastasis and invasion. Moreover, CRK proteins are the major phosphorylation substrates of ABL kinase and its oncogenic mutant BCR-ABL kinase. The interaction between CRK and BCR-ABL plays important roles in chronic myeloid leukemia. Hence, inhibiting the interaction of CRK with BCR-ABL is an attractive way to attenuate cancer metastasis. Herein, we report the development of a peptide inhibitor, PRM-3, targeting the interaction between CRK-II and ABL kinase. PRM-3 binds to the N-terminal SH3 (nSH3) domain in CRK-II with a 10 nM affinity and prevents the interaction between CRK-II and ABL kinase. An in vitro biochemical assay demonstrated that PRM-3 inhibits the ABL-dependent phosphorylation of CRK-II more effectively than imatinib. Remarkably, PRM-3 also inhibited the CRK phosphorylation by T315I-ABL kinase, which is resistant to all first- and second-generation tyrosine kinase inhibitors. Our study provides a promising alternative approach to overcome the drug resistance of ABL kinase.

The Molecular Mechanisms Underlying the Hijack of Host Proteins by the 1918 Spanish Influenza Virus.

The 1918 Spanish influenza A virus (IAV) caused one of the most serious pandemics in history. The nonstructural protein 1 (NS1) of the 1918 IAV hijacks the interaction between human CrkII and JNK1. Little is, however, known about its molecular mechanism. Here, we performed X-ray crystallography, NMR relaxation dispersion experiment, and fluorescence spectroscopy to determine the structural, kinetic, and thermodynamic mechanisms underlying the hijacking of CrkII by 1918 IAV NS1. We observed that the interaction between a proline-rich motif in NS1 and the N-terminal SH3 domain of CrkII displays strikingly rapid kinetics and exceptionally high affinity with 100-fold faster kon and 3300-fold lower Kd compared to those for the CrkII-JNK1 interaction. These results provide molecular insight into the mechanism by which 1918 IAV NS1 hijacks CrkII and disrupts its interactions with critical cellular signaling proteins.

Kinetic Insights into the Binding between the nSH3 Domain of CrkII and Proline-Rich Motifs in cAbl.

The interaction between CrkII and cAbl is implicated in diverse cellular processes. This interaction starts with the binding of the N-terminal Src homology 3 (nSH3) domain of CrkII to the proline-rich motifs of cAbl (PRMscAbl). Despite its critical importance, the detailed binding mechanism between the nSH3 domain and PRMs remains elusive. In this study, we used nuclear magnetic resonance Carr-Purcell-Meiboom-Gill relaxation dispersion experiment to study the binding kinetics between the nSH3 domain of CrkII and PRMscAbl. Our results highlight that the nSH3 domain binds to three PRMscAbl with very high on- and off-rate constants, indicating the transient nature of the binding. To further characterize the binding transition state, we conducted the Eyring and linear free energy relationship analyses using temperature-dependent kinetic data. These data indicate that the binding transition state of the nSH3 domain and PRM is accompanied by small activation enthalpy, owing to partial desolvation of the transition state. These results also highlight the similarity between the transition and free states, in terms of structure and energetics. Although the binding of the nSH3 domain and PRM displays the features consistent with a diffusion-limited process within our experimental conditions, further tests are necessary to determine if the binding is a true diffusion-limited process.

Binding Mechanism of the N-Terminal SH3 Domain of CrkII and Proline-Rich Motifs in cAbl.

The N-terminal Src homology 3 (nSH3) domain of a signaling adaptor protein, CT-10 regulator of kinase II (CrkII), recognizes proline-rich motifs (PRMs) of binding partners, such as cAbl kinase. The interaction between CrkII and cAbl kinase is involved in the regulation of cell spreading, microbial pathogenesis, and cancer metastasis. Here, we report the detailed biophysical characterizations of the interactions between the nSH3 domain of CrkII and PRMs in cAbl. We identified that the nSH3 domain of CrkII binds to three PRMs in cAbl with virtually identical affinities. Structural studies, by using x-ray crystallography and NMR spectroscopy, revealed that the binding modes of all three nSH3:PRM complexes are highly similar to each other. Van 't Hoff analysis revealed that nSH3:PRM interaction is associated with favorable enthalpy and unfavorable entropy change. The combination of experimentally determined thermodynamic parameters, structure-based calculations, and (15)N NMR relaxation analysis highlights the energetic contribution of conformational entropy change upon the complex formation, and water molecules structured in the binding interface of the nSH3:PRM complex. Understanding the molecular basis of nSH3:PRM interaction will provide, to our knowledge, new insights for the rational design of small molecules targeting the interaction between CrkII and cAbl.

Site-selective glycosylation of hemoglobin with variable molecular weight oligosaccharides: potential alternative to PEGylation.

Poly(ethylene glycol) (PEG) conjugation (i.e., PEGylation) is a commonly used strategy to increase the circulatory half-life of therapeutic proteins and colloids; however, few viable alternatives exist to replicate its functions. Herein, we report a method for the rapid site-selective glycosylation of proteins with variously sized carbohydrates, up to a molecular weight (MW) of 10,000, thus serving as a potential alternative for PEGylation. More importantly, the method developed has two unique features. First, traditional protecting group strategies that typically accompany the modification of the carbohydrate fragments are circumvented, allowing for the facile site-selective glycosylation of a desired protein with variously sized glycans. Second, the methodology employed is not limited by oligosaccharide size; consequently, glycans of MW similar to that of PEG, used in the PEGylation of therapeutic proteins, can be employed. To demonstrate the usefulness of this technology, hemoglobin (Hb) was site-selectively glycosylated with a series of carbohydrates of increasing MW (from 504 to ∼10,000). Hb was selected on the basis of the vast wealth of biochemical and biophysical knowledge present in the literature and because of its use as a precursor in the synthesis/formulation of artificial red blood cell substitutes. Following the successful site-selective glycosylation of Hb, the impact of increasing the glycan MW on Hb's biophysical properties was investigated in vitro.

Structure of Greyhound hemoglobin: origin of high oxygen affinity.

This study presents the crystal structure of Greyhound hemoglobin (GrHb) determined to 1.9 Šresolution. GrHb was found to crystallize with an α1ß1 dimer in the asymmetric unit and belongs to the R2 state. Oxygen-affinity measurements combined with the fact that GrHb crystallizes in the R2 state despite the high-salt conditions used for crystallization strongly indicate that GrHb can serve as a model high-oxygen-affinity hemoglobin (Hb) for higher mammals, especially humans. Structural analysis of GrHb and its comparison with the R2-state of human Hb revealed several regions that can potentially contribute to the high oxygen affinity of GrHb and serve to rationalize the additional stability of the R2-state of GrHb. A previously well studied hydrophobic cluster of bar-headed goose Hb near α119 was also incorporated in the comparison between GrHb and human Hb. Finally, a structural comparison with generic dog Hb and maned wolf Hb was conducted, revealing that in contrast to GrHb these structures belong to the R state of Hb and raising the intriguing possibility of an additional allosteric factor co-purifying with GrHb that can modulate its quaternary structure.

Altered architecture of substrate binding region defines the unique specificity of UDP-GalNAc 4-epimerases.

UDP-hexose 4-epimerases play a pivotal role in lipopolysaccharide (LPS) biosynthesis and Leloir pathway. These epimerases are classified into three groups based on whether they recognize nonacetylated UDP-hexoses (Group 1), both N-acetylated and nonacetylated UDP-hexoses (Group 2) or only N-acetylated UDP-hexoses (Group 3). Although the catalysis has been investigated extensively, yet a definitive model rationalizing the substrate specificity of all the three groups on a common platform is largely lacking. In this work, we present the crystal structure of WbgU, a novel UDP-hexose 4-epimerase that belongs to the Group 3. WbgU is involved in biosynthetic pathway of the unusual glycan 2-deoxy-L-altruronic acid that is found in the LPS of the pathogen Pleisomonas shigelloides. A model that defines its substrate specificity is proposed on the basis of the active site architecture. Representatives from all the three groups are then compared to rationalize their substrate specificity. This investigation reveals that the Group 3 active site architecture is markedly different from the "conserved scaffold" of the Group 1 and the Group 2 epimerases and highlights the interactions potentially responsible for the origin of specificity of the Group 3 epimerases toward N-acetylated hexoses. This study provides a platform for further engineering of the UDP-hexose 4-epimerases, leads to a deeper understanding of the LPS biosynthesis and carbohydrate recognition by proteins. It may also have implications in development of novel antibiotics and more economic synthesis of UDP-GalNAc and downstream products such as carbohydrate based vaccines.

Overexpression and topology of bacterial oligosaccharyltransferase PglB.

Campylobacter jejuni contains a post-translational N-glycosylation system in which a STT3 homologue, PglB, functions as the oligosaccharyltransferase. Herein, we established a method for obtaining relatively large quantities of homogenous PglB proteins. PglB was overexpressed in Escherichia coli C43(DE3) at a level of 1 mg/L cell cultures. The activity of purified PglB was verified using a chemically synthesized sugar donor: N-acetylgalactosamine-diphospho-undecaprenyl (GalNAc-PP-Und) and a synthesized peptide acceptor. The result confirms that PglB is solely responsible for the oligosaccharyltransferase activity and complements the finding that PglB exhibits relaxed sugar substrate specificity. In addition, we performed the topology mapping of PglB using the PhoA/LacZ fusion method. The topological model shows that PglB possesses 11 transmembrane segments and two relatively large periplasmic regions other than the C-terminal domain, which is consistent with the proposal of the common N(cyt)-C(peri) topology with 11 transmembrane segments for the STT3 family proteins.

Site-selective glycosylation of hemoglobin on Cys beta93.

In this work, we describe the synthesis and characterization of a novel glycosylated hemoglobin (Hb) with high oxygen affinity as a potential Hb-based oxygen carrier. Site-selective glycosylation of bovine Hb was achieved by conjugating a lactose derivative to Cys 93 on the beta subunit of Hb. LC-MS analysis indicates that the reaction was quantitative, with no unmodified Hb present in the reaction product. The glycosylation site was identified by chymotrypsin digestion of the glycosylated bovine Hb followed with LC-MS/MS and from the X-ray crystal structure of the glycosylated Hb. The chemical conjugation of the lactose derivative at Cys beta93 yields an oxygen carrier with a high oxygen affinity (P(50) of 4.94 mmHg) and low cooperativity coefficient (n) of 1.20. Asymmetric flow field-flow fractionation (AFFFF) coupled with multiangle static light scattering (MASLS) was used to measure the absolute molecular weight of the glycosylated Hb. AFFFF-MASLS analysis indicates that glycosylation of Hb significantly altered the alpha(2)beta(2)-alphabeta equilibrium compared to native Hb. Subsequent X-ray analysis of the glycosylated Hb crystal showed that the covalently linked lactose derivative is sandwiched between the beta(1) and alpha(2) (and hence by symmetry the beta(2) and alpha(1)) subunits of the tetramer, and the interaction between the saccharide and amino acid residues located at the interface is apparently stabilized by hydrogen bonding interactions. The resultant structural analysis of the glycosylated Hb helps to explain the shift in the alpha(2)beta(2)-alphabeta equilibrium in terms of the hydrogen bonding interactions at the beta(1)alpha(2)/beta(2)alpha(1) interface. Taken together, all of these results indicate that it is feasible to site-specifically glycosylate Hb. This work has great potential in developing an oxygen carrier with defined chemistry that can target oxygen delivery to low pO(2) tissues and organs.