Refinement of triclinic lysozyme: II. The method of stereochemically restrained least squares

Ramanadham, M.; Sieker, L.C.; Jensen, L. H.

doi:10.1107/s0108768189009195

Cited by 100 publications

(107 citation statements)

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“…6,127-rcm-lysozyme conformation differs uniquely from native lysozyme at its C terminus. Plots of the main-chain rms deviations per residue are shown for all pairwise comparisons of the two 6,127-rcm-lysozyme forms (rcmA and rcmB) and two crystal forms of native lysozyme [tetragonal (22) and triclinic (29,30)]. For comparisons of 6,127-rcm vs. native forms (Upper Left and Upper Right), deviations for rcmA are plotted above the horizontal axis and, for rcmB, below the axis.…”

Section: Resultsmentioning

confidence: 99%

Crystal structure of a ubiquitin-dependent degradation substrate: a three-disulfide form of lysozyme.

Hill

Johnston

Cohen

1993

Proc. Natl. Acad. Sci. U.S.A.

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Covalent attachment of ubiquitin marks substrates for proteolysis, but features that identify ubiquitination targets such as chicken egg white lysozyme are poorly understood. Recognition of lysozyme first requires reduction of Cys-6 Cys-127, one of its four native disulfide bonds, and Cys-6,Cys-127-carboxymethylated (6,127-rcm) lysozyme can mimic this three-disulfide intermediate. The 6,127-rcm form of lysozyme is known to retain a substantially native-like conformation in solution, and we demonstrate that it is this folded structure that is recognized for ubiquitination. Because native lysozyme is not a substrate, differences between the native and three-disulfide structures must include features responsible for selective ubiquitination. The 1.9-A resolution crystal structure of 6,127-rcm-lysozyme, reported here, affords a view of this ubiquitin-dependent degradation substrate. Two conformers of 6,127-rcm-lysozyme were obtained in the crystal. These differ uniquely from crystal forms of native lysozyme by displacement of the C-terminal residues. The structures suggest that localized unfolding at the C terminus of three-disulfide lysozyme allows the complex of E3a (ubiquitin-protein ligase) and E2 (ubiquitincarrier protein) to bind to a surface that includes Lys-1 and the putative ubiquitination site Lys-13. From this we infer that the N-terminal and internal substrate recognition sites on the E3a-E2 complex are separated by -20 A. Intracellular protein degradation is remarkable for its combination of extreme selectivity and the ability to accommodate an enormous variety of substrates. Protein half-lives in vivo span several orders of magnitude. Moreover, mutations, translational errors, mislocalization, and chemical damage all can lead to polypeptides that are rapidly degraded (1-5). How such proteins are distinguished from their long-lived counterparts is largely unknown. In eukaryotes, a major route for intracellular proteolysis involves covalent modification of protein lysine(s) with the protein ubiquitin (Ub) and subsequent degradation by the 26S Ub-dependent protease complex (6-8). Specificity resides, at least in part, with the Ub-protein ligase that marks a substrate for recognition by the protease. This process is best understood for the E3a ligase from rabbit reticulocytes and the related UBRIencoded enzyme from yeast (9-13).One important E3a recognition determinant is the substrate's N terminus, where only a subset of amino acids is permissive for ubiquitination (9-12). A permissive N terminus is not suffi'cient, however, and the relative orientations and distances that separate lysine ubiquitination sites from

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Section: Resultsmentioning

confidence: 99%

Crystal structure of a ubiquitin-dependent degradation substrate: a three-disulfide form of lysozyme.

Hill

Johnston

Cohen

1993

Proc. Natl. Acad. Sci. U.S.A.

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“…ʈ EGAD, pKa values calculated with a pairwise factorable Generalized-Born electrostatic potential and with side-chain rotamer conformations relaxed by a SCMF algorithm (9). **FDPB, pKa values calculated by using an FDPB electrostatic potential of mean force and discrete backbone and side-chain coordinates from the x-ray crystal structures (25)(26)(27)(28)(29). † † Intrinsic model compound pKa values (8).…”

Section: Prediction Of Solvent-dependent Protein Ionization Constantsmentioning

confidence: 99%

Accurate, conformation-dependent predictions of solvent effects on protein ionization constants

Barth

Alber

Harbury

2007

Proc. Natl. Acad. Sci. U.S.A.

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Predicting how aqueous solvent modulates the conformational transitions and influences the pKa values that regulate the biological functions of biomolecules remains an unsolved challenge. To address this problem, we developed FDPB MF, a rotamer repacking method that exhaustively samples side chain conformational space and rigorously calculates multibody protein-solvent interactions. FDPB MF predicts the effects on pKa values of various solvent exposures, large ionic strength variations, strong energetic couplings, structural reorganizations and sequence mutations. The method achieves high accuracy, with root mean square deviations within 0.3 pH unit of the experimental values measured for turkey ovomucoid third domain, hen lysozyme, Bacillus circulans xylanase, and human and Escherichia coli thioredoxins. FDPB MF provides a faithful, quantitative assessment of electrostatic interactions in biological macromolecules.conformational flexibility ͉ electrostatics ͉ pKa prediction ͉ protein modeling B y strongly interacting with charges, the solvent significantly modulates the electrostatic properties of biomolecular systems. Although variations in pH and ionic strength are responsible for physiologically important conformational transitions (1), quantitatively assessing their role in modulating electrostatic energies is particularly challenging (2). Approaches in which solvent is modeled as a continuum polarizable medium while biomolecules are modeled in explicit molecular detail have become widely used in recent years (3, 4). However, no current method combines broad conformational sampling with a rigorous solvation model that can predict quantitatively and efficiently the effects of solvent on protein energetics and conformations.In principle, molecular dynamics and free-energy simulations monitoring protonation events can model accurately the energetic effects of solvation and conformational relaxation of biomolecules. However, despite recent progress, these techniques often fail to converge in a reasonable amount of computer time and therefore do not provide reliable predictions of thermodynamic properties (5, 6).Rotamer repacking methods sample more efficiently and exhaustively the conformational space of biomolecules. However, these methods require the energy function be decomposed into self energies and interaction energies between pairs of residues (7-10). Consequently, repacking methods cannot model the effects of conformational relaxations involving more than two positions at a time. Many important properties of biomolecular surfaces (i.e., their interface with the solvent and the distribution of bulk ions around them) are not pairwise factorable and depend on the simultaneous knowledge of the conformations of all residues. Approximating these effects by relaxing the protein-solvent interface only at the vicinity of solventexposed charged residues does not improve the prediction of pKa values in proteins (11). Current rotamer repacking methods have also difficulties in assessing accurately and reliably the ...

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“…Before cycles 7 and 23 of the refinement of HCAI-I-, the individual B values of protein and solvent atoms were reset to their average values, as reported by Ramanadham, Sieker & Jensen (1990), in view of the limited quality of the data (Table 1).…”

Section: Refinementmentioning

confidence: 99%

Differences in anionic inhibition of human carbonic anhydrase I revealed from the structures of iodide and gold cyanide inhibitor complexes

Kumar

Kannan²,

Sathyamurthi³

1994

Acta Cryst D

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The crystal structures of two anionic inhibitor complexes of human carbonic anhydrase I (HCAI), namely, HCAI-iodide and HCAI-Au(CN)~, have been refined by the restrained least-squares method at 2.2 and 2 A~ nominal resolution, respectively, with good stereochemistry for the final models. The R values have improved from 30.3 to 16.6% for HCAI-iodide and from 28.8 to 17.1% for HCAIAu(CN)2. The sites of inhibitor binding as elucidated are totally different in the two structures. The iodide anion replaces the zinc-bound H20/OHligand and renders the enzyme inactive. This result confirms that the zinc-bound H20/OH-is the activity-linked group in carbonic anhydrase enzymes. Au(CN)2-binds at a different and new site near the zinc ion, without liganding to the metal. The N atom of Au(CN)2 is within hydrogen-bonding distance of the zinc-bound H20/OH group which shifts by about 0.4 A, away from the zinc ion in relation to its position in the native HCAI. It is proposed that the presence of the inhibitor Au(CN)2-results in a conformational reorientation of the activity-linked group, due to hydrogen-bond formation with the inhibitor, which in turn sterically hinders the binding of the substrate CO2 molecule in the active site, leading to the inhibition of HCAI enzyme.

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Refinement of triclinic lysozyme: II. The method of stereochemically restrained least squares

Cited by 100 publications

References 1 publication

Crystal structure of a ubiquitin-dependent degradation substrate: a three-disulfide form of lysozyme.

Crystal structure of a ubiquitin-dependent degradation substrate: a three-disulfide form of lysozyme.

Accurate, conformation-dependent predictions of solvent effects on protein ionization constants

Differences in anionic inhibition of human carbonic anhydrase I revealed from the structures of iodide and gold cyanide inhibitor complexes

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