Showing papers by "David Eisenberg published in 1994"

Method to identify protein sequences that fold into a known three-dimensional structure

Abstract: A computer-assisted method for identifying protein sequences that fold into a known three-dimensional structure. The method determines three key features of each residue's environment within the structure: (1) the total area of the residue's side-chain that is buried by other protein atoms, inaccessible to solvent; (2) the fraction of the side-chain area that is covered by polar atoms (O, N) or water, and (3) the local secondary structure. Based on these parameters, each residue position is categorized into an environment class. In this manner, a three-dimensional protein structure is converted into a one-dimensional environment string. A 3D structure profile table is then created containing score values that represent the frequency of finding any of the 20 common amino acids structures at each position of the environment string. These frequencies are determined from a database of known protein structures and aligned sequences.

Domain swapping: entangling alliances between proteins.

Abstract: The comparison of monomeric and dimeric diphtheria toxin (DT) reveals a mode for protein association which we call domain swapping. The structure of dimeric DT has been extensively refined against data to 2.0-A resolution and a three-residue loop has been corrected as compared with our published 2.5-A-resolution structure. The monomeric DT structure has also been determined, at 2.3-A resolution. Monomeric DT is a Y-shaped molecule with three domains: catalytic (C), transmembrane (T), and receptor binding (R). Upon freezing in phosphate buffer, DT forms a long-lived, metastable dimer. The protein chain tracing discloses that upon dimerization an unprecedented conformational rearrangement occurs: the entire R domain from each molecule of the dimer is exchanged for the R domain from the other. This involves breaking the noncovalent interactions between the R domain and the C and T domains, rotating the R domain by 180 degrees with atomic movements up to 65 A, and re-forming the same noncovalent interactions between the R domain and the C and T domains of the other chain of the dimer. This conformational transition explains the long life and metastability of the DT dimer. Several other intertwined, dimeric protein structures satisfy our definition of domain swapping and suggest that domain swapping may be the molecular mechanism for evolution of these oligomers and possibly of oligomeric proteins in general.

Refined structure of monomeric diphtheria toxin at 2.3 A resolution.

Abstract: The structure of toxic monomeric diphtheria toxin (DT) was determined at 2.3 A resolution by molecular replacement based on the domain structures in dimeric DT and refined to an R factor of 20.7%. The model consists of 2 monomers in the asymmetric unit (1,046 amino acid residues), including 2 bound adenylyl 3'-5' uridine 3' monophosphate molecules and 396 water molecules. The structures of the 3 domains are virtually identical in monomeric and dimeric DT; however, monomeric DT is compact and globular as compared to the "open" monomer within dimeric DT (Bennett MJ, Choe S, Eisenberg D, 1994b, Protein Sci 3:0000-0000). Detailed differences between monomeric and dimeric DT are described, particularly (1) changes in main-chain conformations of 8 residues acting as a hinge to "open" or "close" the receptor-binding (R) domain, and (2) a possible receptor-docking site, a beta-hairpin loop protruding from the R domain containing residues that bind the cell-surface DT receptor. Based on the monomeric and dimeric DT crystal structures we have determined and the solution studies of others, we present a 5-step structure-based mechanism of intoxication: (1) proteolysis of a disulfide-linked surface loop (residues 186-201) between the catalytic (C) and transmembrane (T) domains; (2) binding of a beta-hairpin loop protruding from the R domain to the DT receptor, leading to receptor-mediated endocytosis; (3) low pH-triggered open monomer formation and exposure of apolar surfaces in the T domain, which insert into the endosomal membrane; (4) translocation of the C domain into the cytosol; and (5) catalysis by the C domain of ADP-ribosylation of elongation factor 2.

An evolutionary approach to folding small alpha-helical proteins that uses sequence information and an empirical guiding fitness function

Abstract: Three short protein sequences have been guided by computer to folds resembling their crystal structures. Initially, peptide fragment conformations ranging in size from 9 to 25 residues were selected from a database of known protein structures. A fragment was selected if it was compatible with a segment of the sequence to be folded, as judged by three-dimensional profile scores. By linking the selected fragment conformations together, hundreds of trial structures were generated of the same length and sequence as the protein to be folded. These starting trial structures were then improved by an evolutionary algorithm. Selection pressure for improving the structures was provided by an energy function that was designed to guide the conformational search procedure toward the correct structure. We find that by evolution of only 400 structures for fewer than 1400 generations, the overall fold of some small helical proteins can be computed from the sequence, with deviations from observed structures of 2.5-4.0 A for C alpha atoms.

Refined structure of dimeric diphtheria toxin at 2.0 Å resolution

Abstract: The refined structure of dimeric diphtheria toxin (DT) at 2.0 A resolution, based on 37,727 unique reflections (F > 1 sigma (F)), yields a final R factor of 19.5% with a model obeying standard geometry. The refined model consists of 523 amino acid residues, 1 molecule of the bound dinucleotide inhibitor adenylyl 3'-5' uridine 3' monophosphate (ApUp), and 405 well-ordered water molecules. The 2.0-A refined model reveals that the binding motif for ApUp includes residues in the catalytic and receptor-binding domains and is different from the Rossmann dinucleotide-binding fold. ApUp is bound in part by a long loop (residues 34-52) that crosses the active site. Several residues in the active site were previously identified as NAD-binding residues. Glu 148, previously identified as playing a catalytic role in ADP-ribosylation of elongation factor 2 by DT, is about 5 A from uracil in ApUp. The trigger for insertion of the transmembrane domain of DT into the endosomal membrane at low pH may involve 3 intradomain and 4 interdomain salt bridges that will be weakened at low pH by protonation of their acidic residues. The refined model also reveals that each molecule in dimeric DT has an "open" structure unlike most globular proteins, which we call an open monomer. Two open monomers interact by "domain swapping" to form a compact, globular dimeric DT structure. The possibility that the open monomer resembles a membrane insertion intermediate is discussed.

Structural model for the reaction mechanism of glutamine synthetase, based on five crystal structures of enzyme-substrate complexes

Abstract: Glutamine synthetase brings nitrogen into metabolism by condensing ammonia and glutamate, with the aid of ATP, to yield glutamine, ADP, and inorganic phosphate. Here we present five crystal structures of GS complexed with each of two substrates, Glu and AMPPNP (an ATP analog), with a transition-state analogue, L-methionine-S-sulfoximine, and with each of two products, Gln and ADP. GS of the present study is from Salmonella typhimurium, has Mn2+ bound, and is fully unadenylylated. Protein-metal-substrate interactions and small but significant conformational changes induced by substrate binding are defined by Fourier maps. On the basis of these maps, we propose a tentative structure-based enzymatic mechanism of glutamine synthesis with these steps: (1) ATP binds first at the top of the funnel-shaped active site cavity, adjacent to the n2 Mn2+; Arg 359 moves toward the Glu binding site. (2) Glu binds adjacent to the n1 Mn2+ at the bottom of the active site near a flexible loop (residues 324-328). As proposed earlier by Meister and others, Glu attacks the gamma-phosphorus atom of ATP to produce gamma-glutamyl phosphate and ADP. (3) The presence of ADP (but not ATP) moves Arg 339 toward the Pi site, perhaps stabilizing the gamma-glutamyl phosphate, and moves Asp 50' of the adjacent subunit toward a putative ammonium ion site, enhancing binding of this third substrate. Deprotonation of the ammonium ion, perhaps by Asp 50', permits the resulting active species, ammonia, to attack the gamma-glutamyl phosphate, forming a tetrahedral intermediate.(ABSTRACT TRUNCATED AT 250 WORDS)

Dynamic transitions of the transmembrane domain of diphtheria toxin: disulfide trapping and fluorescence proximity studies.

Abstract: Translocation of the catalytic domain of diphtheria toxin across the endosomal membrane to the cytosolic compartment depends on low-pH-triggered insertion of the toxin's T (transmembrane) domain into the membrane. The T domain, consisting of nine alpha-helices arranged in three layers, was cloned and expressed as a discrete protein in Escherichia coli, and mutant forms were prepared and characterized. To investigate the relative movements of the three layers under various conditions, we generated two mutant forms of the domain, each containing an artificial intramolecular disulfide bridge linking the buried apolar hairpin (TH8-TH9) to one of the other two layers. Both disulfides inhibited exposure of the domain's apolar regions in solution at low pH, as determined by 2-p-toluidinylnaphthalene-6-sulfonate binding, and blocked its ability to form channels in artificial bilayers. Reduction of the bridges abolished these effects. Reduced forms of the mutant proteins were reacted with pyrenylmaleimide, a fluorescent probe, to monitor separation of the layers. Strong excimer bands seen in both mutants at neutral pH were undiminished at pH 5, indicating the retention of gross conformation in solution under acidic conditions. The addition of phospholipid vesicles at pH 5, but not at pH 7.5, quenched excimer fluorescence, reflecting the physical separation of the TH8-TH9 hairpin from the other layers upon the T domain's interaction with the bilayer. The results indicate that (i) the conformation of the isolated T domain closely resembles that seen in the whole toxin, (ii) the TH8-TH9 hairpin separates from both of the other layers of the domain as an essential step of membrane insertion, and (iii) this separation is triggered by contact of the domain with the membrane under acidic conditions.

Fusion proteins as tools for crystallization: the lactose permease from Escherichia coli.

Abstract: A novel strategy is presented for the crystallization of membrane proteins or other proteins with low solubility and/or stability. The method is illustrated with the lactose permease from Escherichia coli, in which a fusion is constructed between the permease and a 'carrier' protein. The carrier is a soluble, stable protein with its C and N termini close together in space at the surface of the protein, so that the carrier can be introduced into an internal position of the target protein. The carrier is chosen with convenient spectral or enzymatic properties, making the fusion protein easier to handle than the native molecule. Data are presented for the successful construction, expression and purification of a fusion product between lactose permease and cytochrome b(562) from E. coli. The lactose transport activity of the fusion protein is similar to that of wild-type lactose permease, and the fusion product has an absorption spectrum in the visible range which is essentially identical to that of cytochrome b(562). The fusion protein has a higher proportional polar surface area than wild-type permease, and should have better possibilities of forming the strong directional intermolecular contacts required of a crystal lattice.

Interactions of nucleotides with fully unadenylylated glutamine synthetase from Salmonella typhimurium.

Abstract: Glutamine synthetase (GS) catalyzes the ATP-dependent biosynthesis of glutamine from glutamate and ammonia in the presence of divalent cations. To gain insight into the structural basis of the feedback inhibition of GS by AMP, we have studied crystal structures of GS complexes with AMP and the related molecules: AMPPNP (a less hydrolyzable ATP analog), ADP, GDP, adenosine, and adenine. AMP is a feedback inhibitor of GS; ATP and ADP are cofactors, and AMPPNP, GDP, adenosine, and adenine are also GS inhibitors. GS used in this study is from Salmonella typhimurium and is free of covalent modification by adenylylation. All of the crystals examined contain two bound MN2+ ions per GS subunit. The X-ray structures show that all nucleotides bind at the same site, the cofactor ATP binding site, as do adenosine and adenine. Thus from X-ray structures, AMP, adenosine, adenine, and GDP would be expected to inhibit GS-Mn by competing with the substrate ATP for the active site. This suggestion from the crystal structures that AMP is competitive with respect to ATP is supported by kinetic measurements using the biosynthetic assay.

The three-dimensional profile method using residue preference as a continuous function of residue environment.

Abstract: In the 3-dimensional profile method, the compatibility of an amino acid sequence for a given protein structure is scored as the sum of the preferences of the residues for their environments in the 3D structure. In the original method (Bowie JU, Luthy R, Eisenberg D, 1991, Science 253:164-170), residue environments were quantized into 18 discrete environmental classes. Here, amino acid residue preferences are expressed as a continuous function of environmental variables (residue area buried and fractional area buried by polar atoms). This continuous representation of residue preferences, expressed as a Fourier series, avoids the abrupt change of preference of residues in slightly different environments, as encountered in the original method with its 18 discrete environmental classes. When compared with the discrete 18-class representation of residue environments, this continuous 3D profile is found to be more sensitive in identifying sequences that fold into the profiled structure but share with it little sequence identity. The continuous 3D profile is also less sensitive to errors in environmental variables than is the discrete 3D profile. The continuous 3D profile can also be used to detect wrong folds or incorrectly modeled segments in an otherwise correct structure, as could the discrete 3D profile (Luthy R, Bowie JU, Eisenberg D, 1992, Nature 356:83-85). Moreover, the progress of structure improvement during atomic refinement can also be monitored by examining the profile scores in a moving-window scan. Finally, by defining a functional form for profile scores, we open the way to profile atomic refinement in which an atomic structure adjusts to produce residue environments more compatible with the protein side chains.

Structure of the Isolated Catalytic Domain of Diphtheria Toxin

Abstract: The structure of the isolated catalytic domain of diphtheria toxin at pH 5.0 was determined by X-ray crystallography at 2.5 A resolution and refined to an R-factor of 19.7%. The domain is bound to its endogenous inhibitor adenylyl(3'-->5')uridine 3'-monophosphate (ApUp). The structure of this 190-residue domain, which was expressed in and isolated from Escherichia coli, is essentially identical to the structure of the catalytic domain within whole diphtheria toxin determined at pH 7.5. However, there are two adjacent surface loops (loop 66-78 and loop 169-176) that exhibit clear differences when compared to the structure of the catalytic domain in whole diphtheria toxin. Although both loops are at the surface of the protein and are relatively flexible, the chain trace is well-defined in the electron density. The main structural difference is the closer approach of loops 66-78 and 169-176. We ascribe this structural change mainly to the absence of the neighboring transmembrane domain in the isolated catalytic domain as compared to whole diphtheria toxin. We suggest that this change represents the first step of the structural transition from the catalytic domain in whole diphtheria toxin to the translocated form of the domain. The changes are described in detail, and their implications for membrane translocation are discussed.

Crystal structure of the unactivated ribulose 1,5-bisphosphate carboxylase/oxygenase complexed

Abstract: The crystal structure of unactivated ribulose 1,s-bisphosphate carboxylase/oxygenase from Nicotiana rabacum complexed with a transition state analog, 2-carboxy-~-arabinitol 1,s-bisphosphate, was determined to 2.7 A resolution by X-ray crystallography. The transition state analog binds at the active site in an extended conformation. As compared to the binding of the same analog in the activated enzyme, the analog binds in a reverse orientation. The active site Lys 201 is within hydrogen bonding distance of the carboxyl oxygen of the analog. Loop 6 (residues 330-339) remains open and flexible upon binding of the analog in the unactivated enzyme, in contrast to the closed and ordered loop 6 in the activated enzyme complex. The transition state analog is exposed to solvent due to the open conformation of loop 6.

Solid-state phase transition in the crystal structure of ribulose 1,5-bisphosphate carboxylase/oxygenase

Abstract: The crystal structure is described of ribulose 1,5-bisphosphate carboxylase/oxygenase in a new crystal form. This new form (form V) was obtained from a previously known crystal form (form III) through a solid-state phase transition. The solid-state phase transition was brought about by transferring the crystal from a high-salt low-pH mother liquor to a low-salt high-pH synthetic mother liquor. The interplay of electrostatic repulsion and osmotic pressure induced a unit-cell shrinkage of 24 A along the c axis and expansion of 4 A along the a and b axes. The space group also changed from I422 to I4. The new crystal form was shown to be more resistant to X-ray radiation damage, which suggests the effect of crystal stabilization by non-penetrating molecules. The structure of ribulose 1,5-bisphosphate carboxylase/oxygenase in the new crystal form is compared with that of the old crystal form.

Crystallization studies of the human immunodeficiency virus (HIV-1) Tat protein and its trans-activation response element (TAR) RNA

Abstract: Small single crystals are reported of a complex between a small peptide fragment of the HIV-1 Tat protein and a fragment of the RNA to which it binds. Tat is responsible for enhancing the level of expression of the human immunodeficiency virus type 1 (HIV-1) and is a logical target for AIDS therapy. Tat may function to increase the level of transcription initiation or to prevent premature termination of transcripts. In vitro, Tat binds through its basic domain (two Lys and six Arg in nine residues) to a three-nucleotide bulge of a stem-loop RNA structure called TAR. Complex formation between Tat and TAR is necessary for Tat activity. Peptides which contain the basic region of Tat also bind to TAR RNA. We have carried out crystallization experiments on a 27-nucleotide fragment of TAR RNA and on complexes between two Tat peptides and TAR.