A critical view on conservative mutations

Jonson, Per Harald; Petersen, Steffen B.

doi:10.1093/protein/14.6.397

Cited by 31 publications

(33 citation statements)

References 35 publications

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“…Serine residues were inserted because serine was shown to be one of the best candidates for decreasing the mean hydrophobicity without creating steric clashes (20). Similar mutants (with a different mean hydrophobicity compared to the Ebola virus wild-type peptide) were already designed for the SIV tilted peptide (18,26).…”

Section: Figmentioning

confidence: 99%

Distribution of Hydrophobic Residues Is Crucial for the Fusogenic Properties of the Ebola Virus GP2 Fusion Peptide

Adam

Lins

Stroobant

et al. 2004

J Virol

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The lipid-destabilizing properties of the N-terminal domain of the GP2 of Ebola virus were investigated. Our results suggest that the domain of Ebola virus needed for fusion is shorter than that previously reported. The fusogenic properties of this domain are related to its oblique orientation at the lipid/water interface owing to an asymmetric distribution of the hydrophobic residues when helical.The Ebola virus, known for its deadly outbreaks, causes hemorrhagic fevers in humans, yet there is still no effective vaccine or antiviral treatment available.The virion consists of a central ribonucleoprotein core linked by two matrix proteins to a glycoprotein (GP) carrying a lipid bilayer derived from the host cell.The GPs form trimeric spikes on the virion surface and are responsible for receptor binding and fusion of the virus to the host membrane. The GP is cleaved into two parts: GP1 and GP2. The GP2 seems to fold spontaneously into a trimeric, highly ␣-helical, rod-shaped structure, similar to HA2 in influenza virus and to gp41 in human immunodeficiency virus (25). Glycoprotein 2 (GP2) contains a fusion peptide at its N-terminal implicated in virus cell entry. It has previously been demonstrated that the Ebola virus fusion peptide predicted by Gallaher (14), from residues 23 to 38, induces fusion with liposomes and that mutations in this peptide inhibit Ebola virus infectivity (19,32).We have shown that the fusion peptides of simian immunodeficiency virus (SIV) and bovine leukemia virus have an asymmetric distribution of their hydrophobicity residues, the net hydrophobicity increasing from one end of the helix to the other (18,35). This particular distribution is characteristic of the so-called tilted peptides (3,4,5). Recently the orientation of the SIV tilted peptide was determined in planar lipid bilayers by neutron lamellar diffraction. The peptide was shown to form an angle of 55°with the membrane surface when helical, confirming our computational predictions (2). We have previously suggested that the Ebola virus fusion peptide also has such a distribution of hydrophobic residues (34).The present study examines the fusogenic properties of the Ebola virus fusion peptide 25 to 35 in relation to the hydrophobicity gradient by combining modeling and biophysical approaches.We have analyzed the Ebola virus fusion peptide sequence with respect to its hydrophobicity with the hydrophobic cluster analysis. This method is based on an analysis of the shape and position of hydrophobic clusters in a given protein sequence (13). The 25 to 35 fragment (AIGLAWIPYFG) shows a hydrophobicity profile similar to that of the SIV tilted peptide, well known for its fusogenic properties (22, 26), although, as shown on Fig. 1, the two stretches have no sequence identity.The three-dimensional structure of the 25 to 35 Ebola virus peptide was calculated as previously described, assuming a helical secondary structure (3,6,7,28). Small peptides can adopt ␣ and ␤ type structures (12,22,33). It was shown that the ability of the Ebola viru...

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Section: Figmentioning

confidence: 99%

Distribution of Hydrophobic Residues Is Crucial for the Fusogenic Properties of the Ebola Virus GP2 Fusion Peptide

Adam

Lins

Stroobant

et al. 2004

J Virol

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“…Serine was chosen because it is very common in tight protein turns [10], such as loop 2 of PAF where Asp19 is located. Furthermore, it shows a high positive correlation with aspartic acid according to the model of Jonson and Petersen [11], which suggests that a substitution of these amino acid residues exhibits "a high chance of maintaining the thermodynamic stability of the 3D structure". Detailed NMR analyses revealed significant electrostatic surface differences and slight changes in the dynamics at local Ser19 and in the distant loop 3.…”

Section: Introductionmentioning

confidence: 99%

D19S Mutation of the Cationic, Cysteine-Rich Protein PAF: Novel Insights into Its Structural Dynamics, Thermal Unfolding and Antifungal Function

et al. 2017

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The cysteine-rich, cationic, antifungal protein PAF is abundantly secreted into the culture supernatant of the filamentous Ascomycete Penicillium chrysogenum. The five β-strands of PAF form a compact β-barrel that is stabilized by three disulphide bonds. The folding of PAF allows the formation of four surface-exposed loops and distinct charged motifs on the protein surface that might regulate the interaction of PAF with the sensitive target fungus. The growth inhibitory activity of this highly stable protein against opportunistic fungal pathogens provides great potential in antifungal drug research. To understand its mode of action, we started to investigate the surface-exposed loops of PAF and replaced one aspartic acid at position 19 in loop 2 that is potentially involved in PAF active or binding site, with a serine (Asp19 to Ser19). We analysed the overall effects, such as unfolding, electrostatic changes, sporadic conformers and antifungal activity when substituting this specific amino acid to the fairly indifferent amino acid serine. Structural analyses revealed that the overall 3D solution structure is virtually identical with that of PAF. However, PAFD19S showed slightly increased dynamics and significant differences in the surface charge distribution. Thermal unfolding identified PAFD19S to be rather a two-state folder in contrast to the three-state folder PAF. Functional comparison of PAFD19S and PAF revealed that the exchange at residue 19 caused a dramatic loss of antifungal activity: the binding and internalization of PAFD19S by target cells was reduced and the protein failed to trigger an intracellular Ca2+ response, all of which are closely linked to the antifungal toxicity of PAF. We conclude that the negatively charged residue Asp19 in loop 2 is essential for full function of the cationic protein PAF.

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“…Notably, the mutations considered resulted, on average, in negative BLOSUM62 scores, indicating generally nonconservative mutations. 74 These results do not mean that charge, hydrophobicity, volume, and BLOSUM62 scores do not have an effect on the affinity for different cofactors. Instead, they imply that the effect of each factor separately is not monotonic or even discernible in isolation of all other metrics.…”

Section: Analysis Of Results From Previous Cofactor Engineering Studiesmentioning

confidence: 90%

Computational design of Candida boidinii xylose reductase for altered cofactor specificity

et al. 2009

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In this study we introduce a computationally-driven enzyme redesign workflow for altering cofactor specificity from NADPH to NADH. By compiling and comparing data from previous studies involving cofactor switching mutations, we show that their effect cannot be explained as straightforward changes in volume, hydrophobicity, charge, or BLOSUM62 scores of the residues populating the cofactor binding site. Instead, we find that the use of a detailed cofactor binding energy approximation is needed to adequately capture the relative affinity towards different cofactors. The implicit solvation models Generalized Born with molecular volume integration and Generalized Born with simple switching were integrated in the iterative protein redesign and optimization (IPRO) framework to drive the redesign of Candida boidinii xylose reductase (CbXR) to function using the non-native cofactor NADH. We identified 10 variants, out of the 8,000 possible combinations of mutations, that improve the computationally assessed binding affinity for NADH by introducing mutations in the CbXR binding pocket. Experimental testing revealed that seven out of ten possessed significant xylose reductase activity utilizing NADH, with the best experimental design (CbXR-GGD) being 27-fold more active on NADH. The NADPH-dependent activity for eight out of ten predicted designs was either completely abolished or significantly diminished by at least 90%, yielding a greater than 10 4 -fold change in specificity to NADH (CbXR-REG). The remaining two variants (CbXR-RTT and CBXR-EQR) had dual cofactor specificity for both nicotinamide cofactors.

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A critical view on conservative mutations

Cited by 31 publications

References 35 publications

Distribution of Hydrophobic Residues Is Crucial for the Fusogenic Properties of the Ebola Virus GP2 Fusion Peptide

Distribution of Hydrophobic Residues Is Crucial for the Fusogenic Properties of the Ebola Virus GP2 Fusion Peptide

D19S Mutation of the Cationic, Cysteine-Rich Protein PAF: Novel Insights into Its Structural Dynamics, Thermal Unfolding and Antifungal Function

Computational design of Candida boidinii xylose reductase for altered cofactor specificity

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