Structural characteristics of the hydrophobic patch of azurin and its interaction with p53: a site-directed spin labeling study

Site-directed spin labeling (SDSL) is a powerful tool for monitoring protein structure, dynamics and conformational changes. In this study, the domain-specific properties of azurin and its interaction with p53 were studied using this technique. Mutations of six residues, that are located in the hydrophobic patch of azurin, were prepared and spin labeled. Spectra of the six azurin mutants in solution showed that spin labeled residues 45 and 63 are in a very restricted environment, residues 59 and 65 are in a spacious environment and have free movement, and residues 49 and 51 are located in a relatively closed pocket. Polarity experiments confirmed these results. The changes observed in the spectra of spin labeled azurin upon interaction with p53 indicate that the hydrophobic patch is involved in this interaction. Our results provide valuable insight into the topographic structure of the hydrophobic domain of azurin, as well as direct evidence of its interaction with p53 in solution via the hydrophobic patch. Cytotoxicity studies of azurin mutants showed that residues along the hydrophobic patch are important for its cytotoxicity.     

Effects of hesperidin,a flavanone glycoside interaction on the conformation,stability, and aggregation of lysozyme: multispectroscopic and molecular dynamic simulation studies?

Hesperidin (HESP), a flavanone glycoside, shows high antioxidant properties and posses ability to go through the blood–brain barrier. Therefore, it could be a potential drug molecule against aggregation based diseases such as Alzheimer’s, Parkinson’s, and systemic amyloidoses. In this work, we investigated the potential of HESP to interact with hen egg-white lysozyme (HEWL) monomer and prevent its aggregation. The HESP–HEWL binding studies were performed using a fluorescence quenching technique, molecular docking and molecular dynamics simulations. We found a strong interaction of HESP with the lysozyme monomer (K_a, ~ 5 × 10⁴ M^?1) mainly through hydrogen bonding, water bridges, and hydrophobic interactions. We showed that HESP molecule spanned the highly aggregation prone region (amino acid residues 48-101) of HEWL and prevented its fibrillar aggregation. Further, we found that HESP binding completely inhibited amorphous aggregation of the protein induced by disulfide-reducing agent tries-(2-carboxyethyl) phosphine. Conformational and stability studies as followed by various tertiary and secondary structure probes revealed that HESP binding only marginally affected the lysozyme monomer conformation and increased both stability and reversibility of the protein against thermal denaturation. Future studies should investigate detail effects of HESP on solvent dynamics, structure, and toxicity of various aggregates. The answers to these questions will not only target the basic sciences, but also have application in biomedical and biotechnological sciences.     

Hydrophobic patches on the surfaces of protein structures

A survey of hydrophobic patches on the surface of 112 soluble, monomeric proteins is presented. The largest patch on each individual protein averages around 400 Ų but can range from 200 to 1,200 Ų. These areas are not correlated to the sizes of the proteins and only weakly to their apolar surface fraction. Ala, Lys, and Pro have dominating contributions to the apolar surface for smaller patches, while those of the hydrophobic amino acids become more important as the patch size Increases. The hydrophilic amino acids expose an approximately constant fraction of their apolar area independent of patch size; the hydrophobic residue types reach similar exposure only in the larger patches. Though the mobility of residues on the surface is generally higher, it decreases for hydrophilic residues with Increasing patch size. Several characteristics of hydrophobic patches catalogued here should prove useful in the design and engineering of proteins. © 1996 Wiley-Liss, Inc.     

Structural reorganisation and potential toxicity of oligomeric species formed during the assembly of amyloid fibrils

Increasing evidence indicates that oligomeric protein assemblies may represent the molecular species responsible for cytotoxicity in a range of neurological disorders including Alzheimer and Parkinson diseases. We use all-atom computer simulations to reveal that the process of oligomerization can be divided into two steps. The first is characterised by a hydrophobic coalescence resulting in the formation of molten oligomers in which hydrophobic residues are sequestered away from the solvent. In the second step, the oligomers undergo a process of reorganisation driven by interchain hydrogen bonding interactions that induce the formation of β sheet rich assemblies in which hydrophobic groups can become exposed. Our results show that the process of aggregation into either ordered or amorphous species is largely determined by a competition between the hydrophobicity of the amino acid sequence and the tendency of polypeptide chains to form arrays of hydrogen bonds. We discuss how the increase in solvent-exposed hydrophobic surface resulting from such a competition offers an explanation for recent observations concerning the cytotoxicity of oligomeric species formed prior to mature amyloid fibrils.     

Alpha-helix stability in proteins. I. Empirical correlations concerning substitution of side-chains at the N and C-caps and the replacement of alanine by glycine or serine at solvent-exposed surfaces.

The importance of amino acid side-chains in helix stability has been investigated by making a series of mutations at the N-caps, C-caps and internal positions of the solvent-exposed faces of the two alpha-helices of barnase. There is a strong positional and context dependence of the effect of a particular amino acid on stability. Correlations have been found that provide insight into the physical basis of helix stabilization. The relative effects of Ala and Gly (or Ser) may be rationalized on the basis of solvent-accessible surface areas: burial of hydrophobic surface stabilizes the protein as does exposure to solvent of unpaired hydrogen bond donors or acceptors in the protein. There is a good correlation between the relative stabilizing effects of Ala and Gly at internal positions with the total change in solvent-accessible hydrophobic surface area of the folded protein on mutation of Ala----Gly. The relationship may be extended to the N and C-caps by including an extra term in hydrophilic surface area for the solvent exposure of the non-intramolecularly hydrogen-bonded main-chain CO, NH or protein side-chain hydrogen bonding groups. The requirement for solvent exposure of the C-cap main-chain CO groups may account for the strong preference for residues having positive phi and psi angles at this position, since this alpha L-conformation results in the largest solvent exposure of the C-terminal CO groups. Glycine in an alpha L-conformation results in the greatest exposure of these CO groups. Further, the side-chains of His, Asn, Arg and Lys may, with positive phi and psi-angles, form a hydrogen bond with the backbone CO of residue in position C -3 (residues are numbered relative to the C-cap). The preferences at the C-cap are Gly much greater than His greater than Asn greater than Arg greater than Lys greater than Ala approximately Ser approximately greater than Asp. The preferences at the N-cap are determined by hydrogen bonding of side-chains or solvent to the exposed backbone NH groups and are: Thr approximately Asp approximately Ser greater than Gly approximately Asn greater than Gln approximately Glu approximately His greater than Ala greater than Val much greater than Pro. These general trends may be obscured when mutation allows another side-chain to become a surrogate cap.(ABSTRACT TRUNCATED AT 400 WORDS)     

Exploring the differences and similarities between urea and thermally driven denaturation of bovine serum albumin: intermolecular forces and solvation preferences

The interactions of bovine serum albumin (BSA) with urea/water were investigated by computer simulation. It was revealed that the BSA-hydrophobic residues in urea solutions favored contact with urea more than with water. Energy decomposition analysis showed that van der Waals energy was the dominant driving force behind urea affinity for hydrophobic residues, whereas coulombic attraction was largely responsible for water affinity for these residues. Meanwhile, urea–BSA hydrogen bond energies were found to be weaker than water–BSA hydrogen bond energies. The greater strength of water–BSA hydrogen bonds than urea–BSA hydrogen bonds, and the opposing preferential interaction between the BSA and urea suggest that disruption of hydrophobic interaction predominates urea–protein denaturation. In pure water, hydrophobic residues showed aggregation tendencies at 323 K, suggesting an increase in hydrophobicity, while at 353 K the residues were partly denatured due to loss of hydrogen bonds; thus, disruption of hydrophobic interactions appeared to contribute less to thermal denaturation.     

Regulation of the E3 ubiquitin ligase activity of MDM2 by an N-terminal pseudo-substrate motif

The tumor suppressor p53 has evolved a MDM2-dependent feedback loop that promotes p53 protein degradation through the ubiquitin–proteasome system. MDM2 is an E3-RING containing ubiquitin ligase that catalyzes p53 ubiquitination by a dual-site mechanism requiring ligand occupation of its N-terminal hydrophobic pocket, which then stabilizes MDM2 binding to the ubiquitination signal in the DNA-binding domain of p53. A unique pseudo-substrate motif or “lid” in MDM2 is adjacent to its N-terminal hydrophobic pocket, and we have evaluated the effects of the flexible lid on the dual-site ubiquitination reaction mechanism catalyzed by MDM2. Deletion of this pseudo-substrate motif promotes MDM2 protein thermoinstability, indicating that the site can function as a positive regulatory element. Phospho-mimetic mutation in the pseudo-substrate motif at codon 17 (MDM2^S17D) stabilizes the binding of MDM2 towards two distinct peptide docking sites within the p53 tetramer and enhances p53 ubiquitination. Molecular modeling orientates the phospho-mimetic pseudo-substrate motif in equilibrium over a charged surface patch on the MDM2 at Arg⁹⁷/Lys⁹⁸, and mutation of these residues to the MDM4 equivalent reverses the activating effect of the phospho-mimetic mutation on MDM2 function. These data highlight the ability of the pseudo-substrate motif to regulate the allosteric interaction between the N-terminal hydrophobic pocket of MDM2 and its central acidic domain, which stimulates the E3 ubiquitin ligase function of MDM2. This model of MDM2 regulation implicates an as yet undefined lid-kinase as a component of pro-oncogenic pathways that stimulate the E3 ubiquitin ligase function of MDM2 in cells.     

High-resolution structure of an HIV zinc fingerlike domain via a new NMR-based distance geometry approach

A new method is described for determining molecular structures from NMR data. The approach utilizes 2D NOESY back-calculations to generate simulated spectra for structures obtained from distance geometry (DG) computations. Comparison of experimental and back-calculated spectra, including analysis of cross-peak buildup and auto-peak decay with increasing mixing time, provides a quantitative measure of the consistence between the experimental data and generated structures and allows for use of tighter interproton distance constraints. For the first time, the "goodness" of the generated structures is evaluated on the basis of their consistence with the actual experimental data rather than on the basis of consistence with other generated structures. This method is applied to the structure determination of an 18-residue peptide with an amino acid sequence comprising the first zinc fingerlike domain from the gag protein p55 of HIV. This is the first structure determination to atomic resolution for a retroviral zinc fingerlike complex. The peptide [Zn(p55F1)] exhibits a novel folding pattern that includes type I and type II NH-S tight turns and is stabilized both by coordination of the three Cys and one His residues to zinc and by extensive internal hydrogen bonding. The backbone folding is significantly different from that of a "classical" DNA-binding zinc finger. Residues C(1)-F(2)-N(3)-C(4)-G(5)-K(6) fold in a manner virtually identical with the folding observed by X-ray crystallography for related residues in the iron domain of rubredoxin; superposition of all main-chain and Cys side-chain atoms of residues C(1)-K(6) of Zn(p55F1) onto residues C(6)-Y(11) and C(39)-V(44) of rubredoxin gives RMSDs of 0.46 and 0.35 A, respectively. The side chains of conservatively substituted Phe and Ile residues implicated in genomic RNA recognition form a hydrophobic patch on the peptide surface.     

Aprotinin interacts with substrate-binding site of human dipeptidyl peptidase III

Abstract

Human dipeptidyl peptidase III (hDPP III) is a zinc-exopeptidase of the family M49 involved in final steps of intracellular protein degradation and in cytoprotective pathway Keap1-Nrf2. Biochemical and structural properties of this enzyme have been extensively investigated, but the knowledge on its contacts with other proteins is scarce. Previously, polypeptide aprotinin was shown to be a competitive inhibitor of hDPP III hydrolytic activity. In this study, aprotinin was first investigated as a potential substrate of hDPP III, but no degradation products were demonstrated by MALDI-TOF mass spectrometry. Subsequently, molecular details of the protein–protein interaction between aprotinin and hDPP III were studied by molecular modeling. Docking and long molecular dynamics (MD) simulations have shown that aprotinin interacts by its canonical binding epitope with the substrate binding cleft of hDPP III. Thereby, free N-terminus of aprotinin is distant from the active-site zinc. Enzyme-inhibitor complex is stabilized by intermolecular hydrogen bonding network, electrostatic and hydrophobic interactions which mostly involve constituent amino acid residues of the hDPP III substrate binding subsites S1, S1', S2, S2' and S3'. This is the first study that gives insight into aprotinin binding to a metallopeptidase.

Communicated by Ramaswamy H. Sarma     

Mutational analysis and NMR spectroscopy of quail cysteine and glycine-rich protein CRP2 reveal an intrinsic segmental flexibility of LIM domains.

The LIM domain is a conserved cysteine and histidine-containing structural module of two tandemly arranged zinc fingers. It has been identified in single or multiple copies in a variety of regulatory proteins, either in combination with defined functional domains, like homeodomains, or alone, like in the CRP family of LIM proteins. Structural studies of CRP proteins have allowed a detailed evaluation of interactions in LIM-domains at the molecular level. The packing interactions in the hydrophobic core have been identified as a significant contribution to the LIM domain fold, whereas hydrogen bonding within each single zinc binding site stabilizes zinc finger geometry in a so-called "outer" or "indirect" coordination sphere. Here we report the solution structure of a point-mutant of the carboxyl-terminal LIM domain of quail cysteine and glycine-rich protein CRP2, CRP2(LIM2)R122A, and discuss the structural consequences of the disruption of the hydrogen bond formed between the guanidinium side-chain of Arg122 and the zinc-coordinating cysteine thiolate group in the CCHC rubredoxin-knuckle. The structural analysis revealed that the three-dimensional structure of the CCHC zinc binding site in CRP2(LIM2)R122A is adapted as a consequence of the modified hydrogen bonding pattern. Additionally, as a result of the conformational rearrangement of the zinc binding site, the packing interactions in the hydrophobic core region are altered, leading to a change in the relative orientation of the two zinc fingers with a concomitant change in the solvent accessibilities of hydrophobic residues located at the interface of the two modules. The backbone dynamics of residues located in the folded part of CRP2(LIM2)R122A have been characterized by proton-detected(15)N NMR spectroscopy. Analysis of the R2/R1ratios revealed a rotational correlation time of approximately 6.2 ns and tumbling with an axially symmetric diffusion tensor (D parallel/D perpendicular=1.43). The relaxation data were also analyzed using a reduced spectral density mapping approach. As in wild-type CRP2(LIM2), significant mobility on a picosecond/nanosecond time-scale was detected, and conformational exchange on a microsecond time-scale was identified for residues located in loop regions between secondary structure elements. In summary, the relative orientation of the two zinc binding sites and the accessibility of hydrophobic residues is not only determined by hydrophobic interactions, but can also be modified by the formation and/or breakage of hydrogen bonds. This may be important for the molecular interactions of an adaptor-type LIM domain protein in macromolecular complexes, particularly for the modulation of protein-protein interactions.     

Protein stability and surface electrostatics: a charged relationship

Engineering proteins to withstand a broad range of conditions continues to be a coveted objective, holding the potential to advance biomedicine, industry, and our understanding of disease. One way of achieving this goal lies in elucidating the underlying interactions that define protein stability. It has been shown that the hydrophobic effect, hydrogen bonding, and packing interactions between residues in the protein interior are dominant factors that define protein stability. The role of surface residues in protein stability has received much less attention. It has been believed that surface residues are not important for protein stability particularly because their interactions with the solvent should be similar in the native and unfolded states. In the case of surface charged residues, it was sometimes argued that solvent exposure meant that the high dielectric of the solvent will further decrease the strength of the charge-charge interactions. In this paper, we challenge the notion that the surface charged residues are not important for protein stability. We computationally redesigned sequences of five different proteins to optimize the surface charge-charge interactions. All redesigned proteins exhibited a significant increase in stability relative to their parent proteins, as experimentally determined by circular dichroism spectroscopy and differential scanning calorimetry. These results suggest that surface charge-charge interactions are important for protein stability and that rational optimization of charge-charge interactions on the protein surface can be a viable strategy for enhancing protein stability.     

Controlling aggregation propensity in A53T mutant of alpha-synuclein causing Parkinson’s disease

Understanding α-synuclein in terms of fibrillization, aggregation, solubility and stability is fundamental in Parkinson’s disease (PD). The three familial mutations, namely, A30P, E46K and A53T cause PD because the hydrophobic regions in α-synuclein acquire β-sheet configuration, and have a propensity to fibrillize and form amyloids that cause cytotoxicity and neurodegeneration. On simulating the native form and mutants (A30P, E46K and A53T) of α-synuclein in water solvent, clear deviations are observed in comparison to the all-helical 1XQ8 PDB structure. We have identified two crucial residues, ⁴⁰Val and ⁷⁴Val, which play key roles in β-sheet aggregation in the hydrophobic regions 36-41 and 68-78, respectively, leading to fibrillization and amyloidosis in familial (A53T) PD. We have also identified V40D_V74D, a double mutant of A53T (the most amyloidogenic mutant). The simultaneous introduction of these two mutations in A53T nearly ends its aggregation propensity, increases its solubility and positively enhances its thermodynamic stability.     

Reversible and irreversible modifications ofβ-lactoglobulin upon exposure to heat

Modifications in the exposure to the solvent of hydrophobic residues, changes in their organization into surface hydrophobic patches, and alterations in the dimerization equilibrium ofβ-lactoglobulin upon thermal treatment at neutralpH were studied. Exposure of tryptophan residues was temperature dependent and was essentially completed on the time scale of seconds. Reorganization of generic hydrophobic protein patches on the protein surface was monitored through binding of 1,8-anilinonaphthalenesulfonate, and was much slower than changes in tryptophan exposure. Different phases in surface hydrophobicity changes were related to the swelling and the subsequent collapse of the protein, which formed a metastable swollen intermediate. Heat treatment ofβ-lactoglobulin also resulted in the formation of soluble oligomeric aggregates. The aggregation process was studied as a function of temperature, demonstrating that (i) dimer dissociation was a necessary step in a sequential polymerization mechanism and (ii) cohesion of hydrophobic patches was the major driving force for aggregation.     

Effect of Y220C Mutation on p53 and Its Rescue Mechanism: A Computer Chemistry Approach

Mutation causes inactivation of ‘p53’ tumor suppressor protein in almost fifty percent of cancers in humans. Outside the DNA-binding surface of p53, Y220C is the most common cancerous mutation. Previous studies have shown that a surface cavity is created by this mutation which destabilizes p53. PhiKan083, a carbazole derivative capable of binding with that cavity, and slows down its thermal denaturation rate. We investigated, theoretically, on mechanisms of structural stability loss due to Y220C mutation and mechanisms of stability restoration by PhiKan083 at the atomic level. From this study it is found that in Tp53C, Tyr220 has five electrostatic interactions with residues Val 147, Prol51, Pro153 and Pro223 located on S3/S4 loop and S7/S8 loop. The S7/S8 loop is stabilized by these electrostatic interactions. Due to the Y220C mutation all these electrostatic interactions are lost. As a result the structural fluctuation occurs at S7/S8 loop, and the loop is displaced from its original position after 6 ns MD simulation. When PhiKan083 is present (inserted) at the mutation site it provides five electrostatic interactions with Pro155, Glu221 and Thr230, and two hydrogen bonds with Leu145 and Asp228, respectively. These interactions provided by Pkikan083 stabilized the S7/S8 loop, and as a result it couldn’t be displaced. Our results showed that due to Y220C mutation p53 became destabilized through structural fluctuations surrounding the mutation site. When PhiKan083 is present at the Y220C mutation site (in 2vuk), it provides electrostatic and hydrogen bonding interactions among residue-220, its neighboring residues and PhiKan08. These interactions give additional stability to Y220C mutant p53, thus Y220C mutant p53 doesn’t destabilize.     

How Modification of Accessible Lysines to Phenylalanine Modulates the Structural and Functional Properties of Horseradish Peroxidase: A Simulation Study

Horseradish Peroxidase (HRP) is one of the most studied peroxidases and a great number of chemical modifications and genetic manipulations have been carried out on its surface accessible residues to improve its stability and catalytic efficiency necessary for biotechnological applications. Most of the stabilized derivatives of HRP reported to date have involved chemical or genetic modifications of three surface-exposed lysines (K174, K232 and K241). In this computational study, we altered these lysines to phenylalanine residues to model those chemical modifications or genetic manipulations in which these positively charged lysines are converted to aromatic hydrophobic residues. Simulation results implied that upon these substitutions, the protein structure becomes less flexible. Stability gains are likely to be achieved due to the increased number of stable hydrogen bonds, improved heme-protein interactions and more integrated proximal Ca²⁺ binding pocket. We also found a new persistent hydrogen bond between the protein moiety (F174) and the heme prosthetic group as well as two stitching hydrogen bonds between the connecting loops GH and F′F″ in mutated HRP. However, detailed analysis of functionally related structural properties and dynamical features suggests reduced reactivity of the enzyme toward its substrates. Molecular dynamics simulations showed that substitutions narrow the bottle neck entry of peroxide substrate access channel and reduce the surface accessibility of the distal histidine (H42) and heme prosthetic group to the peroxide and aromatic substrates, respectively. Results also demonstrated that the area and volume of the aromatic-substrate binding pocket are significantly decreased upon modifications. Moreover, the hydrophobic patch functioning as a binding site or trap for reducing aromatic substrates is shrunk in mutated enzyme. Together, the results of this simulation study could provide possible structural clues to explain those experimental observations in which the protein stability achieved concurrent with a decrease in enzyme activity, upon manipulation of charge/hydrophobicity balance at the protein surface.     

Ice-binding mechanism of winter flounder antifreeze proteins

We have studied the winter flounder antifreeze protein (AFP) and two of its mutants using molecular dynamics simulation techniques. The simulations were performed under four conditions: in the gas phase, solvated by water, adsorbed on the ice (2021) crystal plane in the gas phase and in aqueous solution. This study provided details of the ice-binding pattern of the winter flounder AFP. Simulation results indicated that the Asp, Asn, and Thr residues in the AFP are important in ice binding and that Asn and Thr as a group bind cooperatively to the ice surface. These ice-binding residues can be collected into four distinct ice-binding regions: Asp-1/Thr-2/Asp-5, Thr-13/Asn-16, Thr-24/Asn-27, and Thr-35/Arg-37. These four regions are 11 residues apart and the repeat distance between them matches the ice lattice constant along the (1102) direction. This match is crucial to ensure that all four groups can interact with the ice surface simultaneously, thereby, enhancing ice binding. These Asx (x = p or n)/Thr regions each form 5-6 hydrogen bonds with the ice surface: Asn forms about three hydrogen bonds with ice molecules located in the step region while Thr forms one to two hydrogen bonds with the ice molecules in the ridge of the (2021) crystal plane. Both the distance between Thr and Asn and the ordering of the two residues are crucial for effective ice binding. The proper sequence is necessary to generate a binding surface that is compatible with the ice surface topology, thus providing a perfect "host/guest" interaction that simultaneously satisfies both hydrogen bonding and van der Waals interactions. The results also show the relation among binding energy, the number of hydrogen bonds, and the activity. The activity is correlated to the binding energy, and in the case of the mutants we have studied the number of hydrogen bonds. The greater the number of the hydrogen bonds the greater the antifreeze activity. The roles van der Waals interactions and the hydrophobic effect play in ice binding are also highlighted. For the latter it is demonstrated that the surface of ice has a clathratelike structure which favors the partitioning of hydrophobic groups to the surface of ice. It is suggested that mutations that involve the deletion of hydrophobic residues (e.g., the Leu residues) will provide insight into the role the hydrophobic effect plays in partitioning these peptides to the surface of ice.     

Aggregation of zinc‐free p53 is inhibited by Hsp90 but not other chaperones

The structured DNA‐binding domain (DBD) of p53 is a well‐known client protein of the chaperone Hsp90. The p53 DBD contains a single zinc ion, coordinated by the side chains of Cys176, His179, Cys238, and Cys242; zinc coordination plays a structural role to stabilize the DBD and is required for its DNA binding. The ambiguous nature of the p53‐Hsp90 interaction, together with the stabilizing role of the zinc in the structure of the DBD, prompted us to examine the interaction of Hsp90 with zinc‐free p53 DBD. NMR spectroscopy and native gel electrophoresis did not show any apparent preference for the interaction of the destabilized zinc‐free form of p53 DBD with Hsp90. Intriguingly, however, at lower protein concentrations, closer to physiological concentrations, the addition of Hsp90, but not other chaperones such as Hsp70, Hsp40, p23, and HOP, appears to slow or prevent the aggregation of zinc‐free p53 DBD. This result suggests that part of the function of the Hsp90‐p53 interaction in the cell may be to stabilize the apoprotein in the absence of zinc.     

The essential functions of NEDD8 are mediated via distinct surface regions, and not by polyneddylation in Schizosaccharomyces pombe

The ubiquitin-like protein NEDD8 is highly conserved in eukaryotes, from man to Schizosaccharomyces pombe. NEDD8 conjugation to cullin proteins is a prerequisite for cullin based E3 ubiquitin ligase activity, and essential for S. pombe viability. Here, we have performed alanine scanning mutagenesis of all conserved surface residues and show that the majority of essential residues were located around the hydrophobic patch and the C-terminus. However, we further identified essential residues not previously reported to be involved in ubiquitin ligase regulation that importantly do not prevent Ned8p conjugation. We also find that mutation of all conserved lysine residues in Ned8p, did not affect yeast viability, suggesting that mono-neddylation is sufficient for yeast viability under most conditions.