Mutational analysis of the leucine zipper motif in the Newcastle disease virus fusion protein.

The paramyxovirus fusion proteins have a highly conserved leucine zipper motif immediately upstream from the transmembrane domain of the F1 subunit (R. Buckland and F. Wild, Nature [London] 338:547, 1989). To determine the role of the conserved leucines in the oligomeric structure and biological activity of the Newcastle disease virus (NDV) fusion protein, the heptadic leucines at amino acids 481, 488, and 495 were changed individually and in combination to an alanine residue. While single amino acid changes had little effect on fusion, substitution of two or three leucine residues abolished the fusogenic activity of the protein, although cell surface expression of the mutants was higher than that of the wild-type protein. Substitution of all three leucine residues with alanine did not alter the size of the fusion protein oligomer as determined by sedimentation in sucrose gradients. Furthermore, deletion of the C-terminal 91 amino acids, including the leucine zipper motif and transmembrane domain, resulted in secretion of an oligomeric polypeptide. These results indicate that the conserved leucines are not necessary for oligomer formation but are required for the fusogenic ability of the protein. When the polar face of the potential alpha helix was altered by nonconservative changes of serine to alanine (position 473), glutamic acid to lysine or alanine (position 482), asparagine to lysine (position 485), or aspartic acid to alanine (position 489), the fusogenic ability of the protein was not significantly disrupted. In addition, a double mutant (E482A,D489A) which removed negative charges along one side of the helix had negligible effects on fusion activity.     

Human interleukin 4. The solution structure of a four-helix bundle protein.

Heteronuclear 13C and 15N three-dimensional nuclear magnetic resonance (n.m.r.) techniques have been used to determine the solution structure of human interleukin 4, a four-helix bundle protein. A dynamical simulated annealing protocol was used to calculate an ensemble of structures from an n.m.r. data set of 1735 distance restraints, 101 phi angle restraints and 27 pairs of hydrogen bond restraints. The protein structure has a left-handed up-up-down-down topology for the four helices with the two long overhand loops in the structure being connected by a short section of irregular antiparallel beta-sheet. Analysis of the side-chains in the protein shows a clustering of hydrophobic residues, particularly leucines, in the core of the bundle with the side-chains of charged residues being located on the protein surface. The solution structure has been compared with a recent structure prediction for human interleukin 4 and with crystal structures of other helix bundle proteins.     

Atomic-resolution crystal structure of Borrelia burgdorferi outer surface protein A via surface engineering

Outer surface protein A (OspA) from Borrelia burgdorferi has an unusual dumbbell-shaped structure in which two globular domains are connected with a "single-layer" beta-sheet (SLB). The protein is highly soluble, and it has been recalcitrant to crystallization. Only OspA complexes with Fab fragments have been successfully crystallized. OspA contains a large number of Lys and Glu residues, and these "high entropy" residues may disfavor crystal packing because some of them would need to be immobilized in forming a crystal lattice. We rationally designed a total of 13 surface mutations in which Lys and Glu residues were replaced with Ala or Ser. We successfully crystallized the mutant OspA without a bound Fab fragment and extended structure analysis to a 1.15 Angstroms resolution. The new high-resolution structure revealed a unique backbone hydration pattern of the SLB segment in which water molecules fill the "weak spots" on both faces of the antiparallel beta-sheet. These well-defined water molecules provide additional structural links between adjacent beta-strands, and thus they may be important for maintaining the rigidity of the SLB that inherently lacks tight packing afforded by a hydrophobic core. The structure also revealed new information on the side-chain dynamics and on a solvent-accessible cavity in the core of the C-terminal globular domain. This work demonstrates the utility of extensive surface mutation in crystallizing recalcitrant proteins and dramatically improving the resolution of crystal structures, and provides new insights into the stabilization mechanism of OspA.     

Structural details of ribonuclease H from Escherichia coli as refined to an atomic resolution.

The crystal structure of RNase H from Escherichia coli has been determined by the multiple isomorphous replacement method, and refined by the stereochemically restrained least-squares procedure to a crystallographic R-factor of 0.196 at 1.48 A resolution. In the final structure, the root-mean-square (r.m.s.) deviation for bond lengths is 0.017 A, and for angle distances 0.036 A. The structure is composed of a five-stranded beta-sheet and five alpha-helices, and reveals the details of hydrogen bonding, electrostatic and hydrophobic interactions between intra- and intermolecular residues. The refined structure allows an explanation of the particular interactions between the basic protrusion, consisting of helix alpha III and the following loop, and the remaining major domain. The beta-sheet, alpha II, alpha III and alpha IV form a central hydrophobic cleft that contains all six tryptophan residues, and presumably serves to fix the orientation of the basic protrusion. Two parallel adjacent helices, alpha I and alpha IV, are associated with a few triads of hydrophobic interactions, including many leucine residues, that are similar to the repeated leucine motif. The well-defined electron density map allows detailed discussion of amino acid residues likely to be involved in binding a DNA/RNA hybrid, and construction of a putative model of the enzyme complexed with a DNA/RNA hybrid oligomer. In this model, a protein region, from the Mg(2+)-binding site to the basic protrusion, covers roughly two turns of a DNA/RNA hybrid double helix. A segment (11-23) containing six glycine residues forms a long loop between the beta A and beta B strands. This loop, which protrudes into the solvent region, lies on the interface between the enzyme and a DNA/RNA hybrid in the model of the complex. The mean temperature factors of main-chain atoms show remarkably high values in helix alpha III that constitutes the basic protrusion, suggesting some correlation between its flexibility and the nucleic acid binding function. The Mg(2+)-binding site, surrounded by four invariant acidic residues, can now be described more precisely in conjunction with the catalytic activity. The arrangement of molecules within the crystal appears to be dominated by the cancelling out of a remarkably biased charge distribution on the molecular surface, which is derived in particular from the separation between the acidic Mg(2+)-binding site and the basic protrusion.     

Three-dimensional structure of a human pancreatic ribonuclease variant, a step forward in the design of cytotoxic ribonucleases

We have determined the crystal structure of a human pancreatic ribonuclease or RNase 1 variant at 1.65 A resolution. Five residues in the N-terminal region were substituted by the corresponding amino acids of the bovine seminal RNase. In addition, a Pro to Ser mutation was present at position 50. The substitution of part of the N terminus has been critical both in improving the expression of this enzyme as a recombinant protein and in achieving its crystallisation. The determination of the crystal structure revealed the characteristic RNase fold including a V-shaped beta-sheet and three alpha-helices. It differs from its bovine RNase orthologue mainly in the loop regions. The active-site cleft shows a similar architecture to that of its bovine counterpart, with the essential residues occupying equivalent positions. In the present structure, however, His119 is displaced as it is in the structure of RNase A at high pH. An interaction model of human ribonuclease with the ribonuclease inhibitor, together with inhibition assays, indicate that, in contrast to RNase A, the modification of the loop beta4beta5 is not enough to avoid inhibition. This study represents the first crystallographic approach to the human enzyme, and should constitute an invaluable tool for the design of ribonuclease variants with acquired cytotoxic properties.     

Structure-based substitutions for increased solubility of a designed protein

Manipulation of protein solubility is important for many aspects of protein design and engineering. Previously, we designed a series of consensus ankyrin repeat proteins containing one, two, three and four identical repeats (1ANK, 2ANK, 3ANK and 4ANK). These proteins, particularly 4ANK, are intended for use as a universal scaffold on which specific binding sites can be constructed. Despite being well folded and extremely stable, 4ANK is soluble only under acidic conditions. Designing interactions with naturally occurring proteins requires the designed protein to be soluble at physiological pH. Substitution of six leucines with arginine on exposed hydrophobic patches on the surface of 4ANK resulted in increased solubility over a large pH range. Study of the pH dependence of stability demonstrated that 4ANK is one of the most stable ankyrin repeat proteins known. In addition, analogous leucine to arginine substitutions on the surface of 2ANK allowed the partially folded protein to assume a fully folded conformation. Our studies indicate that replacement of surface-exposed hydrophobic residues with positively charged residues can significantly improve protein solubility at physiological pH.     

Primary structure of a ribonuclease from bovine brain

The primary structure of a pyrimidine base-specific ribonuclease from bovine brain was determined. The sequence determined is (sequence; see text). Although the sequence homology of this RNase with bovine pancreatic RNase A is 78.2%, it consists of 140 amino acid residues, and it is 16 amino acid residues longer than RNase A at the carboxyl-terminal. In addition to an N-glycosylated long carbohydrate chain, the bovine brain RNase has two short O-glycosylated carbohydrate chains at the 129th and the 133rd serine residues. The additional C-terminal tail of the bovine brain RNase has a unique composition: 6 proline, 5 hydrophobic amino acids, and two basic amino acids, arginine and histidine.     

Dimerization of leucine zippers analyzed by random selection.

The leucine zipper is a coiled coil that mediates specific dimerization of bZIP DNA-binding domains. A hydrophobic spine involving the conserved leucines runs down the coiled-coil and is thought to stabilize the dimer. We used the method of random selection to further define the primary sequence requirements for homodimer formation and heterodimer formation with Fos. When positions on either side of the hydrophobic spine of GCN4 are diversified to include the corresponding residues of Jun, a large percentage of the resulting sequences form homodimers, and a large percentage form heterodimers with Fos. Basic residues were preferred, but not essential, at position e of zippers which heterodimerize with Fos. When random sequences containing 5 heptad repeat of leucines are subject to a selection for homodimer formation, a diverse set of sequences is isolated. Certain residues are preferred at each position in the heptad repeat, although no essential primary sequence determinants could be identified. No pair of residues not involving the conserved leucines could be identified which strongly promotes homodimerization. These results suggest that factors determining leucine zipper dimerization are complex, with numerous interactions contributing to the association.     

Protein crystallization by design: chymotrypsinogen without precipitants

Protein crystals are usually obtained by an empirical approach based on extensive screening to identify suitable crystallization conditions. In contrast, we have used a systematic predictive procedure to produce data-quality crystals of bovine chymotrypsinogen A and used them to obtain a refined X-ray structure to 3 A resolution. Measurements of the osmotic second virial coefficient of chymotrypsinogen solutions were used to identify suitable solvent conditions, following which crystals were grown for approximately 30 hours by ultracentrifugal crystallization, without the use of any precipitants. Existing structures of chymotrypsinogen were obtained in solutions including 10-30 % ethanol, whereas simple buffered NaCl solutions were used here. The protein crystallized in the tetragonal space group P4(1)2(1)2, with one molecule per asymmetric unit. The quality of the refined map was very high throughout, with the main-chain atoms of all but four residues clearly defined and with nearly all side-chains also defined. Although only minor differences are seen compared to the structures previously reported, they indicate the possibility of structural changes due to the crystallization conditions used in those studies. Our results show that more systematic crystallization of proteins is possible, and that the procedure can expand the range of conditions under which crystals can be grown successfully and can make new crystal forms available.     

Crystal structure of a hybrid between ribonuclease A and bovine seminal ribonuclease--the basic surface, at 2.0 A resolution.

A variant of bovine pancreatic ribonuclease A has been prepared with seven amino acid substitutions (Q55K, N62K, A64T, Y76K, S80R, E111G, N113K). These substitutions recreate in RNase A the basic surface found in bovine seminal RNase, a homologue of pancreatic RNase that diverged some 35 million years ago. Substitution of a portion of this basic surface (positions 55, 62, 64, 111 and 113) enhances the immunosuppressive activity of the RNase variant, activity found in native seminal RNase, while substitution of another portion (positions 76 and 80) attenuates the activity. Further, introduction of Gly at position 111 has been shown to increase the catalytic activity of RNase against double-stranded RNA. The variant and the wild-type (recombinant) protein were crystallized and their structures determined to a resolution of 2.0 A. Each of the mutated amino acids is seen in the electron density map. The main change observed in the mutant structure compared with the wild-type is the region encompassing residues 16-22, where the structure is more disordered. This loop is the region where the polypeptide chain of RNase A is cleaved by subtilisin to form RNase S, and undergoes conformational change to allow residues 1-20 of the RNase to swap between subunits in the covalent seminal RNase dimer.     

Atomic force microscopy of crystalline insulins: the influence of sequence variation on crystallization and interfacial structure.

The self-association of proteins is influenced by amino acid sequence, molecular conformation, and the presence of molecular additives. In the presence of phenolic additives, LysB28ProB29 insulin, in which the C-terminal prolyl and lysyl residues of wild-type human insulin have been inverted, can be crystallized into forms resembling those of wild-type insulins in which the protein exists as zinc-complexed hexamers organized into well-defined layers. We describe herein tapping-mode atomic force microscopy (TMAFM) studies of single crystals of rhombohedral (R3) LysB28ProB29 that reveal the influence of sequence variation on hexamer-hexamer association at the surface of actively growing crystals. Molecular scale lattice images of these crystals were acquired in situ under growth conditions, enabling simultaneous identification of the rhombohedral LysB28ProB29 crystal form, its orientation, and its dynamic growth characteristics. The ability to obtain crystallographic parameters on multiple crystal faces with TMAFM confirmed that bovine and porcine insulins grown under these conditions crystallized into the same space group as LysB28ProB29 (R3), enabling direct comparison of crystal growth behavior and the influence of sequence variation. Real-time TMAFM revealed hexamer vacancies on the (001) terraces of LysB28ProB29, and more rounded dislocation noses and larger terrace widths for actively growing screw dislocations compared to wild-type bovine and porcine insulin crystals under identical conditions. This behavior is consistent with weaker interhexamer attachment energies for LysB28ProB29 at active growth sites. Comparison of the single crystal x-ray structures of wild-type insulins and LysB28ProB29 suggests that differences in protein conformation at the hexamer-hexamer interface and accompanying changes in interhexamer bonding are responsible for this behavior. These studies demonstrate that subtle changes in molecular conformation due to a single sequence inversion in a region critical for insulin self-association can have a significant effect on the crystallization of proteins.     

Moving the pH Gate of the Kir1.1 Inward Rectifier Channel

Both structural and functional studies suggest that pH gating of the inward rectifier potassium (K) channel, Kir1.1 (ROMK), is mediated by the convergence of 4 hydrophobic leucines (one from each subunit) near the cytoplasmic bundle-crossing of the inner transmembrane helices. We tested this hypothesis by moving the putative leucine gate from the L160-Kir1.1b to other positions along the inner transmembrane helix, and measuring inward current and conductance as functions of internal pH, using the Xenopus oocyte heterologous expression system. Results of these studies indicated that it was possible to replace the putative inward rectifier pH gate at L160-Kir1.1b by either a leucine or methionine at 157-Kir1.1b (G157L-L160G or G157M-L160G). Although both leucine and methionine gated the channel at 157-Kir1.1b, residues of similar hydrophobicity (tyrosine and valine) did not. Hence, hydrophobicity was a necessary but not a sufficient condition for steric gating at 157. This was in contrast to the 160-Kir1.1b locus, where side-chain hydrophobicity was both a necessary and sufficient property for steric gating. Homology models were constructed for all mutants that expressed significant whole-cell currents, using the closed-state coordinates of the prokaryotic inward rectifier, KirBac1.1. Models of mutants that retained pH gating were too narrow at the bundle crossing to permit hydrated K ion permeation in the closed-state. On the other hand, mutants that lost pH gating had ample space at the bundle crossing for hydrated K permeation in the closed-state. These results support our hypothesis that hydrophobic leucines at the cytoplasmic end of the inner transmembrane helices comprise the principal pH gate of Kir1.1, a gate that can be relocated from 160-Kir1.1b to 157-Kir1.1b.     

Crystal packing in six crystal forms of pancreatic ribonuclease.

We compare the molecular packing of bovine pancreatic ribonuclease A (RNase A) in six crystal forms, two grown with alcohol, three with high salt and one with polyethylene glycol as a precipitant. The six packings differ in the number of molecules in contact and in the extent of the contacts, which bury 1570 A2 to 2790 A2 of the RNase surface. Regions of the protein surface involved in the six packings cover almost the whole RNase molecule. The abundance of polar interactions, about one per 200 A2, is the same in all types of precipitants. All molecule-to-molecule contacts are different in the six crystal forms, except for the one that forms a RNase dimer. The dimer has a large interface covering 1800 A2 and eight to ten polar interactions. Its presence in the three salt-grown crystal forms suggests that it is an intermediate in salt induced crystallization. In contrast, the two alcohol-grown forms contain only small interfaces, implying a different mechanism of nucleation.     

NMR structure and molecular dynamics of the in-plane membrane anchor of nonstructural protein 5A from bovine viral diarrhea virus

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a monotopic membrane protein anchored to the membrane by an N-terminal in-plane amphipathic alpha-helix. This membrane anchor is essential for the assembly of a functional viral replication complex. Although amino acid sequences differ considerably, putative membrane anchors with amphipathic features were predicted in NS5A from related Flaviviridae family members, in particular bovine viral diarrhea virus (BVDV), the prototype representative of the genus Pestivirus. We report here the NMR structure of the membrane anchor 1-28 of NS5A from BVDV in the presence of different membrane mimetic media. This anchor includes a long amphipathic alpha-helix of 21 residues interacting in-plane with the membrane interface and including a putative flexible region. Molecular dynamic simulation at a water-dodecane interface used to mimic the surface separating a lipid bilayer and an aqueous medium demonstrated the stability of the helix orientation and the location at the hydrophobic-hydrophilic interface. The flexible region of the helix appears to be required to allow the most favorable interaction of hydrophobic and hydrophilic side chain residues with their respective environment at the membrane interface. Despite the lack of amino acid sequence similarity, this amphipathic helix shares common structural features with that of the HCV counterpart, including a stable, hydrophobic N-terminal segment separated from the more hydrophilic C-terminal segment by a local, flexible region. These structural conservations point toward conserved roles of the N-terminal in-plane membrane anchors of NS5A in replication complex formation of HCV, BVDV, and other related viruses.     

Determinants of the t peptide involved in folding, degradation, and secretion of acetylcholinesterase

The C-terminal 40-residue t peptide of acetylcholinesterase (AChE) forms an amphiphilic alpha helix with a cluster of seven aromatic residues. It allows oligomerization and induces a partial degradation of AChE subunits through the endoplasmic reticulum-associated degradation pathway. We show that the t peptide induces the misfolding of a fraction of AChE subunits, even when mutations disorganized the cluster of aromatic residues or when these residues were replaced by leucines, indicating that this effect is due to hydrophobic residues. Mutations in the aromatic-rich region affected the cellular fate of AChE in a similar manner, with or without mutations that prevented dimerization. Degradation was decreased and secretion was increased when aromatic residues were replaced by leucines, and the opposite occurred when the amphiphilic alpha helix was disorganized. The last two residues (Asp-Leu) somewhat resembled an endoplasmic reticulum retention signal and caused a partial retention but only in mutants possessing aromatic residues in their t peptide. Our results suggested that several "signals" in the catalytic domain and in the t peptide act cooperatively for AChE quality control.     

Mechanism for multiple-substrates recognition of alpha-aminoadipate aminotransferase from Thermus thermophilus

Alpha-aminoadipate aminotransferase (AAA-AT), a homolog of mammalian kynurenine aminotransferase II (Kat II), transfers an amino group to 2-oxoadipate to yield alpha-aminoadipate in lysine biosynthesis through the alpha-aminoadipate pathway in Thermus thermophilus. AAA-AT catalyzes transamination against various substrates, including AAA, glutamate, leucine, and aromatic amino acids. To elucidate the structural change for recognition of various substrates, we determined crystal structures of AAA-AT in four forms: with pyridoxal 5'-phosphate (PLP) (PLP complex), with PLP and leucine (PLP/Leu complex), with N-phosphopyridoxyl-leucine (PPL) (PPL complex), and with N-phosphopyridoxyl-alpha-aminoadipate (PPA) at 2.67, 2.26, 1.75, and 1.67 A resolution, respectively. The PLP complex is in an open state, whereas PLP/Leu, PPL, and PPA complexes are in closed states with maximal displacement (over 7 A) of the alpha2 helix and the beta1 strand in the small domain to cover the active site, indicating that conformational change is induced by substrate binding. In PPL and PLP/Leu complexes, several hydrophobic residues on the alpha2 helix recognize the hydrophobic side chain of the bound leucine moiety whereas, in the PPA complex, the alpha2 helix rotates to place the guanidium moiety of Arg23 on the helix at the appropriate position to interact with the carboxyl side chain of the AAA moiety. These results indicate that AAA-AT can recognize various kinds of substrates using the mobile alpha2 helix. The crystal structures and site-directed mutagenesis revealed that intersubunit-electrostatic interactions contribute to the elevated thermostability of this enzyme.     

Effects of the stabilization of the molten globule state on the folding mechanism of alpha-lactalbumin: a study of a chimera of bovine and human alpha-lactalbumin

The N-terminal half of the alpha-domain (residues 1 to 34) is more important for the stability of the acid-induced molten globule state of alpha-lactalbumin than the C-terminal half (residues 86 to 123). The refolding and unfolding kinetics of a chimera, in which the amino acid sequence of residues 1 to 34 was from human alpha-lactalbumin and the remainder of the sequence from bovine alpha-lactalbumin, were studied by stopped-flow tryptophan fluorescence spectroscopy. The chimeric protein refolded and unfolded substantially faster than bovine alpha-lactalbumin. The stability of the molten globule state formed by the chimera was greater than that of bovine alpha-lactalbumin, and the hydrophobic surface area buried inside of the molecule in the molten globule state was increased by the substitution of residues 1 to 34. Peptide fragments corresponding to the A- and B-helix of the chimera showed higher helix propensity than those of the bovine protein, indicating the contribution of local interactions to the high stability of the molten globule state of the chimera. Moreover, the substitution of residues 1-34 decreased the free energy level of the transition state and increased hydrophobic surface area buried inside of the molecule in the transition state. Our results indicate that local interactions as well as hydrophobic interactions formed in the molten globule state are important in guiding the subsequent structural formation of alpha-lactalbumin.     

Binding of mitochondrial leader sequences to Tom20 assessed using a bacterial two-hybrid system shows that hydrophobic interactions are essential and that some mutated leaders that do not bind Tom20 can still be imported

Previous studies pointed to the importance of leucine residues in the binding of mitochondrial leader sequences to Tom20, an outer membrane protein translocator that initially binds the leader during import. A bacteria two-hybrid assay was here employed to determine if this could be an alternative way to investigate the binding of leader to the receptor. Leucine to alanine and arginine to glutamine mutations were made in the leader sequence from rat liver aldehyde dehydrogenase (pALDH). The leucine residues in the C-terminal of pALDH leader were found to be essential for TOM20 binding. The hydrophobic residues of another mitochondrial leader F1beta-ATPase that were important for Tom20 binding were found at the C-terminus of the leader. In contrast, it was the leucines in the N-terminus of the leader of ornithine transcarbamylase that were essential for binding. Modeling the peptides to the structure of Tom20 showed that the hydrophobic residues from the three proteins could all fit into the hydrophobic binding pocket. The mutants of pALDH that did not bind to Tom20 were still imported in vivo in transformed HeLa cells or in vitro into isolated mitochondria. In contrast, the mutant from pOTC was imported less well ( approximately 50%) while the mutant from F1beta-ATPase was not imported to any measurable extent. Binding to Tom20 might not be a prerequisite for import; however, it also is possible that import can occur even if binding to a receptor component is poor, so long as the leader binds tightly to another component of the translocator.