Determining the three-dimensional fold of a protein from approximate constraints: a simulation study.

We propose a new approach for calculating the three-dimensional (3D) structure of a protein from distance and dihedral angle constraints derived from experimental data. We suggest that such constraints can be obtained from experiments such as tritium planigraphy, chemical or enzymatic cleavage of the polypeptide chain, paramagnetic perturbation of nuclear magnetic resonance (NMR) spectra, measurement of hydrogen-exchange rates, mutational studies, mass spectrometry, and electron paramagnetic resonance. These can be supplemented with constraints from theoretical prediction of secondary structures and of buried/exposed residues. We report here distance geometry calculations to generate the structures of a test protein Staphylococcal nuclease (STN), and the HIV-1 rev protein (REV) of unknown structure. From the available 3D atomic coordinates of STN, we set up simulated data sets consisting of varying number and quality of constraints, and used our group's Self Correcting Distance Geometry (SECODG) program DIAMOD to generate structures. We could generate the correct tertiary fold from qualitative (approximate) as well as precise distance constraints. The root mean square deviations of backbone atoms from the native structure were in the range of 2.0 A to 8.3 A, depending on the number of constraints used. We could also generate the correct fold starting from a subset of atoms that are on the surface and those that are buried. When we used data sets containing a small fraction of incorrect distance constraints, the SECODG technique was able to detect and correct them. In the case of REV, we used a combination of constraints obtained from mutagenic data and structure predictions. DIAMOD generated helix-loop-helix models, which, after four self-correcting cycles, populated one family exclusively. The features of the energy-minimized model are consistent with the available data on REV-RNA interaction. Our method could thus be an attractive alternative for calculating protein 3D structures, especially in cases where the traditional methods of X-ray crystallography and multidimensional NMR spectroscopy have been unsuccessful.

Homology modeling and characterization of IgE binding epitopes of mountain cedar allergen Jun a 3.

The Jun a 3 protein from mountain cedar (Juniperus ashei) pollen, a member of group 5 of the family of plant pathogenesis-related proteins (PR-proteins), reacts with serum IgE from patients with cedar hypersensitivity. We used the crystal structures of two other proteins of this group, thaumatin and an antifungal protein from tobacco, both approximately 50% identical in sequence to Jun a 3, as templates to build homology models for the allergen. The in-house programs EXDIS and FANTOM were used to extract distance and dihedral angle constraints from the Protein Data Bank files and determine energy-minimized structures. The mean backbone deviations for the energy-refined model structures from either of the templates is <1 A, their conformational energies are low, and their stereochemical properties (determined with PROCHECK) are acceptable. The circular dichroism spectrum of Jun a 3 is consistent with the postulated beta-sheet core. Tryptic fragments of Jun a 3 that reacted with IgE from allergic patients all mapped to one helical/loop surface of the models. The Jun a 3 models have features common to aerosol allergens from completely different protein families, suggesting that tertiary structural elements may mediate the triggering of an allergic response.

Mutational analysis of apolipoprotein B mRNA editing enzyme (APOBEC1). structure-function relationships of RNA editing and dimerization.

APOBEC1 is the catalytic subunit of an enzyme complex that mediates apolipoprotein (apo) B mRNA editing. It dimerizes in vitro and requires complementation factor(s) for its editing activity. We have performed a systematic analysis of the structure-functional relationship of APOBEC1 by targeted mutagenesis of various sequence motifs within the protein. Using in vitro RNA editing assay, we found that basic amino acid clusters at the amino-terminal region R15R16R17 and R33K34, are essential for apoB mRNA editing. Mutation of R15R16R17 to K15K16K17 and mutation of R33K34 simultaneously to A33A34 almost completely abolished in vitro editing activity. The carboxy-terminal region of APOBEC1 contains a leucine-rich motif. Deletion analysis of this region indicates that residues 181 to 210 are important for in vitro apoB mRNA editing. Single amino acid substitutions demonstrate that L182, I185, and L189 are important residues required for normal editing function. Furthermore, the double mutant P190A/P191A also lost >90% of editing activity which suggests that a beta turn in this region of the molecule may be essential for proper functioning of APOBEC1. It was suggested that dimerization of APOBEC1 creates an active structure for deamination of apoB mRNA. When we examined the dimerization potential of truncated APOBEC1s using both amino and carboxy termini deletion mutants, we found that amino-terminal deletions up to residue A117 did not impair dimerization activity whereas carboxy-terminal deletions showed diminished dimerization. The systematic and extensive mutagenesis experiments in this study provide information on the role of various sequence motifs identified in APOBEC1 in enzyme catalysis and dimerization.

Template-based docking of a prolactin receptor proline-rich motif octapeptide to FKBP12: implications for cytokine receptor signaling.

A conserved proline-rich motif (PRM) in the cytoplasmic domain of cytokine receptors has been suggested to be a signaling switch regulated by the action of the FK506 binding protein (FKBP) family of peptidylprolyl isomerases (O'Neal KD, Yu-Lee LY, Shearer WT, 1995, Ann NY Acad Sci 766:282-284). We have docked the prolactin receptor PRM (Ile1-Phe2-Pro3-Pro4-Val5-Pro6-Gly7-Pro8) to the ligand binding site of FKBP12. The procedure involved conformational search restricted by NMR restraints (O'Neal KD et al., 1996, Biochem J 315:833-844), energy minimization of the octapeptide conformers so obtained, template-based docking of a selected conformer to FKBP12, and energy refinement of the resulting complex. The template used was the crystal structure of a cyclic FK506-peptide hybrid bound to FKBP12. Val5-Pro6 of the PRM was taken to be the biologically relevant Xaa-Pro bond. The docked conformer is stabilized by two intramolecular hydrogen bonds, N7H7-->O4 and N2H2-->O8, and two intermolecular ones, Ile56; N-H-->O = C:Pro6 and Tyr82:O-H-->O = C:Gly7. This conformer features a Type I beta-turn and has extensive hydrophobic contacts with the FKBP12 binding surface. The observed interactions support the hypothesis that FKBP12 catalyzes cis-trans isomerization in the PRM when it is part of the longer cytoplasmic domain of a cytokine receptor, and suggest a significant role for the PRM in signal transduction.

Structure-activity relationship of quaternary ammonium ions at the external tetraethylammonium binding site of cloned potassium channels.

Changes in the chemical structure of the tetraethylammonium (TEA) ion reduce binding affinity at the external TEA receptor of outwardly rectifying potassium channels. To study the mechanism of selective binding, we applied a variety of hydrophilic quaternary ammonium (QA) ions to the noninactivating mutant of Shaker B T449Y, to Kv3.1, and to Kv3.1 mutants, expressed in Xenopus oocytes. In outside-out patches, QA ions in which ethyl groups of TEA were replaced by methyl groups had a lower affinity than TEA, whereas changes in binding affinity were minor when propyl groups were substituted for ethyl groups. All channels tested showed this pattern. Changes in free energy of binding correlated well with changes in the computed free energy of hydration of the TEA derivatives that we used. The affinity for TEA derivatives was reduced in Kv3.1Y407T, which is in support of the hypothesis that cation pi-electron interaction is involved. Binding affinities of QA ions were higher in Kv3.1 Y407F than in the wild-type, suggesting that the hydroxyl groups of the tyrosines reduce QA binding. The rank order of potency of the QA ions toward the different channels studied was the same. These results indicate that external QA ions bind strongly to hydrophobic pi-electron-rich functions. The selectivity, however, is determined by the physical properties of the QA ion.

The selectivity of different external binding sites for quaternary ammonium ions in cloned potassium channels.

Tetraethylammonium (TEA) is thought to be the most effective quaternary ammonium (QA) ion blocker at the external site of K+ channels, and small changes to the TEA ion reduce its potency. To examine the properties of the external QA receptor, we applied a variety of QA ions to excised patches from human embryonic kidney cells or Xenopus oocytes transfected with the delayed rectifying K+ channels Kv 2.1 and Kv 3.1. In outside-out patches of Kv 3.1, the relative potencies were TEA > tetrapropylammonium (TPA) > tetrabutylammonium (TBA). In contrast to Kv 3.1, the relative potencies in Kv 2.1 were TBA > TEA > TPA. In Kv 3.1 and Kv 2.1, external tetrapentylammonium (TPeA) blocked K+ currents in a fast, reversible and, in contrast to TEA, time-dependent manner. The external binding of TPeA appeared to be voltage independent, unlike the effects of TPeA applied to inside-out patches. External n-alkyl-triethylammonium compounds (C8, C10 chain length) had a lower affinity than TEA in Kv 3.1, but a higher affinity than TEA in Kv 2.1. In Kv 3.1, the decrease in QA affinity was large when one or two methyl groups were substituted for ethyl groups in TEA, but minor when propyl groups replaced ethyl groups. Changes in the free energy of binding could be correlated to changes in the free energy of hydration of TEA derivatives calculated by continuum methodology. These results reveal a substantial hydrophobic component of external QA ion binding to Kv 2.1, and to a lesser degree to Kv 3.1, in addition to the generally accepted electrostatic interactions. The chain length of hydrophobic TEA derivatives affects the affinity for the hydrophobic binding site, whereas the hydropathy of QA ions determines the electrostatic interaction energy.

Secondary structure prediction of the H5 pore of potassium channels.

The 'H5' segment located between the putative fifth and sixth transmembrane helices is the most highly conserved region in voltage-gated potassium channels and it is believed to constitute a major part of the ion conduction path (pore). Here we present a two-step procedure, comprising secondary structure prediction and hydrophobic moment profiling, to predict the structure of this important region. Combined results from the application of the procedure to the H5 region of four voltage-gated and five other K+ channel sequences lead to the prediction of a beta-strand-turn-beta-strand structure for H5. The reasons for the application of these soluble protein methods to parts of membrane proteins are: (i) that pore-lining residues are accessible to water and (ii) that a large enough database of high-resolution membrane protein structures does not yet exist. The results are compared with experimental results, in particular spectroscopic studies of two peptides based on the H5 sequence of SHAKER potassium channel. The procedure developed here may be applicable to water-accessible regions of other membrane proteins.

Molecular dynamics analysis of a ribonuclease C-peptide analogue.

We have carried out a nanosecond molecular dynamics simulation of an analogue of the ribonuclease C-peptide in water. The overall conformation has an extended region for the first three amino acids connected to an alpha-helix for residues 4-13, and this basic structure is preserved throughout the simulation, with helical hydrogen bonds present 87% of the time, on average. The final helical hydrogen bond is spontaneously broken and re-formed several times, providing a detailed picture of such winding/unwinding events. The simulation was used to estimate the effects of internal motion on proton nuclear Overhauser effect spectroscopy (NOESY) intensities for several classes of important cross peaks. Within the helical regions, the effects of internal motion vary only a little from one residue to another for backbone-backbone cross peaks, and the relevant correlation functions reach plateau values within about 50 ps. The spectral simulations show, however, that it may be difficult to establish a close quantitative connection between NOESY cross-peak volumes and measures of helical content.

Conformational studies on beta-bend containing a cis peptide unit.

Conformational studies have been carried out on the X-cis-Pro tripeptide system (a system of three linked peptide units, in the trans-cis-trans configuration) using energy minimization techniques. For X, residues Gly, L-Ala, D-Ala and L-Pro have been used. The energy minima have been classified into different groups based upon the conformational similarity. There are 15, 20, 18 and 6 minima that are possible for the four cases respectively and these fall into 11 different groups. A study of these minima shows that, (i) some minima contain hydrogen bonds--either 4-->1 or 1-->2 type, (ii) the low energy minima qualify themselves as bend conformations, (iii) cis' and trans' conformations are possible for the prolyl residue as also the C gamma-endo and C gamma-exo puckerings, and (iv) for Pro-cis-Pro, cis' at the first prolyl residue is ruled out, due to the high energy. The available crystal structure data on proteins and peptides, containing cis-Pro segment have been examined with a view to find the minima that occur in solid state. The data from protein show that they fall under two groups. The conformation at X in X-cis-Pro is near extended when it is a non-glycyl residue. In both peptides and proteins there exists a preference for trans' conformation at prolyl residue over cis' when X is a non-glycyl residue. The minima obtained can be useful in modelling studies.

Unfolding of an alpha-helix in water.

We describe a 1 ns molecular dynamics simulation of an 18-residue peptide (corresponding to a portion of the H helix of myoglobin) in water. The initial helical conformation progressively frays to a more disordered structure, with the loss of internal secondary structure generally proceeding from the C-terminus toward the N-terminus. Although a variety of mechanisms are involved in the breaking of helical hydrogen bonds, the formation of transient turn structures, with i----i + 3 hydrogen bonds, and bifurcated hydrogen-bond structures intermediate between alpha and turn or 3(10) structures is a common motif. In some cases a single water molecule is inserted into an internal hydrogen bond, but it is also common to have several water molecules involved in transient intermediates. Overall, the results provide new information about the detailed mechanisms by which helices are made and broken in aqueous solution.

Three-dimensional solution structure of a single zinc finger DNA-binding domain.

The three-dimensional solution structure of a zinc finger nucleic acid binding motif has been determined by nuclear magnetic resonance (NMR) spectroscopy. Spectra of a synthetic peptide corresponding to a single zinc finger from the Xenopus protein Xfin yielded distance and dihedral angle constraints that were used to generate structures from distance geometry and restrained molecular dynamics calculations. The zinc finger is an independently folded domain with a compact globular structure in which the zinc atom is bound by two cysteine and two histidine ligands. The polypeptide backbone fold consists of a well-defined helix, starting as alpha and ending as 3(10) helix, packed against two beta strands that are arranged in a hairpin structure. A high density of basic and polar amino acid side chains on the exposed face of the helix are probably involved in DNA binding.

A novel supersecondary structure in globular proteins comprising the collagen-like helix and beta-turn.

A structure consisting of the polyproline-II or collagen-like helix immediately succeeded by a beta-turn is seen in several synthetic peptides and has been suggested to be the conformational requirement for proline hydroxylation in nascent procollagen. Using a simple algorithm for detecting secondary structures, we have analysed crystal structure data on 40 globular proteins and have found eight examples of the collagen-helix + beta-turn supersecondary structure in 15 proteins that contain the collagen-like helical segments.

Occurrence of a single helix of the collagen type in globular proteins.

The occurrence of an eight-residue long segment of polypeptide chain in collagen helical conformation has been detected in bacteriochlorophyll a-protein by the application of an algorithm for identifying secondary structures in globular proteins from their alpha-carbon positions. This segment spans residues 277 to 284 of the protein and is the longest known stretch of collagen helix to be observed in globular proteins.

Identification of secondary structures in globular proteins--a new algorithm.

A new algorithm has been developed for identifying helices, extended structures, and bends from the positions of the alpha-carbon atoms using the virtual bond approach. The parameters used are two virtual bond angles (delta 1 and delta 2), the virtual dihedral angle (theta), and the distance (D) between the terminal alpha-carbon atoms of the tripeptide. The criteria for classification have been worked out by model building as well as from proteins whose complete secondary structures are known. These criteria are as follows: (i) magnitude of theta less than or equal to 60 degrees and (delta 1 + delta 2) less than or equal to 230 degrees for a bend, (ii) for a helix, successive thetas should not differ by more than 30 degrees, and (iii) for an extended structure, the cumulative deviation of the above parameters should not vary by more than 20% from the ideal extended chain. The method developed has been applied successfully to three proteins wherein the coordinates of alpha-carbon atoms alone are known and a complete mapping of the secondary structures has now been obtained. One interesting observation is that the percentage of residues not taking part in helices, extended structures, and bends is very small--of the order of 4%.

Reversals of polypeptide chain in globular proteins.

A simple algorithm has been developed to detect beta-bends and 'loops'-chain reversals containing five amino acid residues, using only coordinates of C alpha-atoms from crystal structure data of globular proteins using the above algorithm. Analysis of bends have showed that the total number of bends in each protein (TB) is linearly related to total number of non-hydrophobic residues in that protein which in turn is related linearly to total number of amino acid residues. Secondly, we found that a large number of consecutive bends occur in each protein which give rise to on an average only three independent residues per turn. Positional preference of amino acid residues in chain reversals is stressed. Consideration of pairs of amino acid residues in positions (i + 1) and (i + 2) of bends seems to provide a more reliable basis for predicting chain reversals in proteins.