CoCUN, a Novel Ubiquitin Binding Domain Identified in N4BP1

Ubiquitin binding domains (UBDs) are modular elements that bind non-covalently to ubiquitin and act as downstream effectors and amplifiers of the ubiquitination signal. With few exceptions, UBDs recognize the hydrophobic path centered on Ile44, including residues Leu8, Ile44, His68, and Val70. A variety of different orientations, which can be attributed to specific contacts between each UBD and surface residues surrounding the hydrophobic patch, specify how each class of UBD specifically contacts ubiquitin. Here, we describe the structural model of a novel ubiquitin-binding domain that we identified in NEDD4 binding protein 1 (N4BP1). By performing protein sequence analysis, mutagenesis, and nuclear magnetic resonance (NMR) spectroscopy of the 15N isotopically labeled protein, we demonstrate that a Phe-Pro motif in N4BP1 recognizes the canonical hydrophobic patch of ubiquitin. This recognition mode resembles the molecular mechanism evolved in the coupling of ubiquitin conjugation to endoplasmic-reticulum (ER) degradation (CUE) domain family, where an invariant proline, usually following a phenylalanine, is required for ubiquitin binding. Interestingly, this novel UBD, which is not evolutionary related to CUE domains, shares a 40% identity and 47% similarity with cullin binding domain associating with NEDD8 (CUBAN), a protein module that also recognizes the ubiquitin-like NEDD8. Based on these features, we dubbed the region spanning the C-terminal 50 residues of N4BP1 the CoCUN domain, for Cousin of CUBAN. By performing circular dichroism and 15N NMR chemical shift perturbation of N4BP1 in complex with ubiquitin, we demonstrate that the CoCUN domain lacks the NEDD8 binding properties observed in CUBAN. We also show that, in addition to mediating the interaction with ubiquitin and ubiquitinated substrates, both CUBAN and CoCUN are poly-ubiquitinated in cells. The structural and the functional characterization of this novel UBD can contribute to a deeper understanding of the molecular mechanisms governing N4BP1 function, providing at the same time a valuable tool for clarifying how the discrimination between ubiquitin and the highly related NEDD8 is achieved

Structural characterization of the Redox-Dependent differences in the Cytochrome P450cam-Putidaredoxin Complex using solution NMR spectroscopy

Complexation between proteins as part of biological electron transfer reactions is driven by precise interactions that are often characterized by short lifetimes, weak affinities and high turnover rates. These complex interactions are difficult to study structurally in physiologically relevant oxidation states due to their transient nature and/or large molecular sizes. One such protein complex in the cytochrome P450 family of enzymes that is of great interest to researchers due to its prototypical nature is the Putidaredoxin (Pdx)- cytochrome P450cam (CYP101) electron transfer complex that is involved in hydroxylation of D-camphor in the bacterium Pseudomonas putida. While the individual protein structures for Pdx and CYP101 have been known for several years in both oxidized and reduced states, high-resolution structural information for the Pdx-CYP101 complex is still lacking in either oxidation state. This structural information is critical to not only determine the electron transfer pathway between the two proteins in this complex, but also to explain the role of Pdx as an effector in substrate turnover. In this study, a solution NMR approach utilizing long-range distance restraints derived from paramagnetic relaxation effects is used to obtain structures of the Pdx-CYP101 complex in both substrate-bound oxidized and a catalytically competent reduced form. Key redox-dependent structural and dynamic differences between the two complexes have been characterized which provide insights into the mechanism of effector activity of Pdx

The development of biomolecular Raman optical activity spectroscopy

Following its first observation over 40 years ago, Raman optical activity (ROA), which may be measured as a small difference in the intensity of vibrational Raman scattering from chiral molecules in right- and left-circularly polarized incident light or, equivalently, the intensity of a small circularly polarized component in the scattered light using incident light of fixed polarization, has evolved into a powerful chiroptical spectroscopy for studying a large range of biomolecules in aqueous solution. The long and tortuous path leading to the first observations of ROA in biomolecules in 1989, in which the author was closely involved from the very beginning, is documented, followed by a survey of subsequent developments and applications up to the present day. Among other things, ROA provides information about motif and fold, as well as secondary structure, of proteins; solution structure of carbohydrates; polypeptide and carbohydrate structure of intact glycoproteins; new insight into structural elements present in unfolded protein sequences; and protein and nucleic acid structure of intact viruses. Quantum chemical simulations of observed Raman optical activity spectra provide the complete three-dimensional structure, together with information about conformational dynamics, of smaller biomolecules. Biomolecular ROA measurements are now routine thanks to a commercial instrument based on a novel design becoming available in 2004

Structural studies of a consensus sequence peptide (CSP) ABAB of apolipoproteins through NMR spectroscopy

Thesis (Ph.D.)--Boston UniversityThe apolipoproteins play critical roles in lipid transport, lipid metabolism and the pathophysiology of dyslipoproteinemias, most importantly atherosclerosis. ApoA-1 is a representative member of the family of exchangeable apolipoproteins and the major apolipoprotein of high density lipoprotein (HDL). HDL is responsible for the pathway of reverse cholesterol transport and the only particle capable of removing cholesterol from peripheral cells for transport to the liver. The sequences ofthe exchangeable apolipoproteins contain 11/22 residue tandem sequence repeats forming amphipathic α-helices that are believed to be responsible for lipid binding. The consensus sequence peptide (CSP) for this repeat was derived based on the characteristic residue distribution of the exchangeable apolipoproteins. The derived consensus sequence containing motifs A, (PLAEELRARLR), and B, (AQLEELRERLG), represent an idealized lipid binding model and fundamental structural motif of the exchangeable apolipoproteins. The recombinant CSP-ABAB peptide was successfully expressed in E. coli and purified. Circular dichroism showed that CSP-ABAB is ~62% α-helical, i.e.~27 residues of 44 residues are in helical conformation. The CSP-ABAB peptide was successfully 15N, 13C labeled and the detailed tertiary structure was explored by NMR spectroscopy. The peptide's backbone and side-chain resonances were successfully assigned and ten water refined structural conformers of CSP-ABAB were generated. The ten structural conformers all employ anti-parallel helical conformation in solution. Hydrophobic inter-helical interactions play a major role to stabilize the antiparallel helical hairpin conformation. There are also intra-/inter-helical salt bridges present on the surface of the CSP-ABAB molecule providing additional stabilization. The structural features of the NMR structures suggest a lipid binding model of CSP-ABAB. When lipids are introduced, the exposed hydrophobic ridge contributed by the twelve leucine residues firstly bind to the lipids. At the same time, a hydrophobic concave surface created by the four alanine residues at the center of the interface is accessed by the introduced lipids. These two steps open the anti-parallel helical hairpin conformation to form a fully extended α-helix. Similar hydrophobic inter-helical stabilization interactions and new intra-/inter-helical salt bridges between two different CSP-ABAB molecules are reformed to stabilize the 'double-belt' arrangement. This lipid binding model of CSP-ABAB sheds light on the lipid binding of apoA-I and the mechanism of HDL formation

NMR studies of metalloproteins

Metalloproteins represent a large share of the proteomes, with the intrinsic metal ions providing catalytic, regulatory, and structural roles critical to protein functions. Structural characterization of metalloproteins and identification of metal coordination features including numbers and types of ligands and metal-ligand geometry, and mapping the structural and dynamic changes upon metal binding are significant for understanding biological functions of metalloproteins. NMR spectroscopy has long been used as an invaluable tool for structure and dynamic studies of macromolecules. Here we focus on the application of NMR spectroscopy in characterization of metalloproteins, including structural studies and identification of metal coordination spheres by hetero-/homo-nuclear metal NMR spectroscopy. Paramagnetic NMR as well as (13)C directly detected protonless NMR spectroscopy will also be addressed for application to paramagnetic metalloproteins. Moreover, these techniques offer great potential for studies of other non-metal binding macromolecules.postprin