Structure of a stabilizing disulfide bridge mutant that closes the active-site cleft of T4 lysozyme.

The engineered disulfide bridge between residues 21 and 142 of phage T4 lysozyme spans the active-site cleft and can be used as a switch to control the activity of the enzyme (Matsumura, M. & Matthews, B.W., 1989, Science 243, 792-794). In the oxidized form the disulfide increases the melting temperature of the protein by 11 degrees C at pH 2. The crystal structure of this mutant lysozyme has been determined in both the reduced and oxidized forms. In the reduced form, the crystal structure of the mutant is shown to be extremely similar to that of wild type. In the oxidized form, however, the formation of the disulfide bridge causes the alpha-carbons of Cys 21 and Cys 142, on opposite sides of the active-site cleft, to move toward each other by 2.5 A. In association with this movement, the amino-terminal domain of the protein undergoes a rigid-body rotation of 5.1 degrees relative to the carboxy-terminal domain. This rotation occurs about an axis passing through the junction of the amino-terminal and carboxy-terminal domains and is also close to the axis that best fits the apparent thermal motion of the amino-terminal domain seen previously in crystals of wild-type lysozyme. Even though the engineered Cys 21-Cys 142 disulfide links together the amino-terminal and carboxy-terminal domains of T4 lysozyme, it does not reduce the apparent mobility of the one domain relative to the other. The pronounced "hinge-bending" mobility of the amino-terminal domain that is suggested by the crystallographic thermal parameters of wild-type lysozyme persists in the oxidized (and reduced) mutant structures. In the immediate vicinity of the introduced disulfide bridge the mutant structure is more mobile (or disordered) than wild type, so much so that the exact conformation of Cys 21 remains obscure. As with the previously described disulfide bridge between residues 9 and 164 of T4 lysozyme (Pjura, P.E., Matsumura, M., Wozniak, J.A., & Matthews, B.W., 1990, Biochemistry 29, 2592-2598), the engineered cross-link substantially enhances the stability of the protein without making the folded structure more rigid.     

Multiple alanine replacements within alpha-helix 126-134 of T4 lysozyme have independent, additive effects on both structure and stability.

In a systematic attempt to identify residues important in the folding and stability of T4 lysozyme, five amino acids within alpha-helix 126-134 were substituted by alanine, either singly or in selected combinations. Together with three alanines already present in the wild-type structure this provided a set of mutant proteins with up to eight alanines in sequence. All the variants behaved normally, suggesting that the majority of residues in the alpha-helix are nonessential for the folding of T4 lysozyme. Of the five individual alanine substitutions it is inferred that four result in slightly increased protein stability and one, the replacement of a buried leucine with alanine, substantially decreased stability. The results support the idea that alanine is a residue of high helix propensity. The change in protein stability observed for each of the multiple mutants is approximately equal to the sum of the energies associated with each of the constituent substitutions. All of the variants could be crystallized isomorphously with wild-type lysozyme, and, with one trivial exception, their structures were determined at high resolution. Substitution of the largely solvent-exposed residues Asp 127, Glu 128, and Val 131 with alanine caused essentially no change in structure except at the immediate site of replacement. Substitutions of the partially buried Asn 132 and the buried Leu 133 with alanine were associated with modest (< or = 0.4 A) structural adjustments. The structural changes seen in the multiple mutants were essentially a combination of those seen in the constituent single replacements. The different replacements therefore act essentially independently not only so far as changes in energy are concerned but also in their effect on structure. The destabilizing replacement Leu 133-->Ala made alpha-helix 126-134 somewhat less regular. Incorporation of additional alanine replacements tended to make the helix more uniform. For the penta-alanine variant a distinct change occurred in a crystal-packing contact, and the "hinge-bending angle" between the amino- and carboxy-terminal domains changed by 3.6 degrees. This tends to confirm that such hinge-bending in T4 lysozyme is a low-energy conformational change.     

Investigation of Domain Motions in Bacteriophage T4 Lysozyme

Abstract

Hinge-bending in T4 lysozyme has been inferred from single amino acid mutant crystalline allomorphs by Matthews and coworkers. This raises an important question: are the different conformers in the unit cell artifacts of crystal packing forces, or do they represent different solution state structures? The objective of this theoretical study is to determine whether domain motions and hinge-bending could be simulated in T4 lysozyme using molecular dynamics. An analysis of a 400 ps molecular dynamics simulation of the 164 amino acid enzyme T4 lysozyme is presented. Molecular dynamics calculations were computed using the Discover software package (Biosym Technologies). All hydrogen atoms were modeled explicitly with the inclusion of all 152 crystallographic waters at a temperature of 300 K. The native T4 lysozyme molecular dynamics simulation demonstrated hinge-bending in the protein. Relative domain motions between the N-terminal and C-terminal domains were evident. The enzyme hinge bending sites resulted from small changes in backbone atom conformations over several residues rather than rotation about a single bound. Two hinge loci were found in the simulation. One locus comprises residues 8–14 near the C-terminal of the A helix; the other site, residues 77–83 near the C-terminal of the C helix. Comparison of several snapshot structures from the dynamics trajectory clearly illustrates domain motions between the two lysozyme lobes. Time correlated atomic motions in the protein were analyzed using a dynamical cross-correlation map. We found a high degree of correlated atomic motions in each of the domains and, to a lesser extent, anticorrelated motions between the two domains. We also found that the hairpin loop in the N-terminal lobe (residues 19–24) acted as a mobile ‘flap’ and exhibited highly correlated dynamic motions across the cleft of the active site, especially with residue 142.     

Systematic mutation of bacteriophage T4 lysozyme

Amber mutations were introduced into every codon (except the initiating AUG) of the bacteriophage T4 lysozyme gene. The amber alleles were introduced into a bacteriophage P22 hybrid, called P22 e416, in which the normal P22 lysozyme gene is replaced by its T4 homologue, and which consequently depends upon T4 lysozyme for its ability to form a plaque. The resulting amber mutants were tested for plaque formation on amber suppressor strains of Salmonella typhimurium. Experiments with other hybrid phages engineered to produce different amounts of wild-type T4 lysozyme have shown that, to score as deleterious, a mutation must reduce lysozyme activity to less than 3% of that produced by wild-type P22 e416. Plating the collection of amber mutants covering 163 of the 164 codons of T4 lysozyme, on 13 suppressor strains that each insert a different amino acid substitutions at every position in the protein (except the first). Of the resulting 2015 single amino acid substitutions in T4 lysozyme, 328 were found to be sufficiently deleterious to inhibit plaque formation. More than half (55%) of the positions in the protein tolerated all substitutions examined. Among (N-terminal) amber fragments, only those of 161 or more residues are active. The effects of many of the deleterious substitutions are interpretable in light of the known structure of T4 lysozyme. Residues in the molecule that are refractory to replacements generally have solvent-inaccessible side-chains; the catalytic Glu11 and Asp20 residues are notable exceptions. Especially sensitive sites include residues involved in buried salt bridges near the catalytic site (Asp10, Arg145 and Arg148) and a few others that may have critical structural roles (Gly30, Trp138 and Tyr161).     

Structural studies of mutants of T4 lysozyme that alter hydrophobic stabilization

Multiple replacements at amino acid position 3 of bacteriophage T4 lysozyme have shown that the conformational stability of the protein is directly governed by the hydrophobicity of the residue substituted (Matsumura, M., Becktel, W. J., and Matthews, B. W. (1988) Nature 334, 406-410). Of the 13 mutant lysozymes made by site-directed mutagenesis, two variants, one with valine (I3V) and the other with tyrosine (I3Y), were crystallized and their structures solved. In this report we describe the crystal structures of these variants at 1.7 A resolution. While the structure of the I3V mutant is essentially the same as that of wild-type lysozyme, the I3Y mutant has substantial changes in its structure. The most significant of these are that the side chain of the tyrosine is not accommodated within the interior of the protein and the amino-terminal polypeptide (residues 1-9) moves 0.6-1.1 A relative to the wild-type structure. Using coordinates based on the wild-type and available mutant structures, solvent accessible surface area of residue 3 as well as the adjacent 9 residues in the folded form were calculated. The free energy of stabilization based on the transfer of these residues from a fully extended form to the interior to the folded protein was found to correlate well with the protein stability determined by thermodynamic analysis. The enhanced thermostability of the variant Ile-3----Leu, relative to wild-type lysozyme, can also be rationalized by surface-area calculations based on a model-built structure. Noncrystallization of most lysozyme variants at position 3 appears to be due to disruption of intermolecular contacts in the crystal. The Ile-3----Val variant is closely isomorphous with wild-type and maintains the same crystal contacts. In the Ile-3----Tyr variant, however, a new set of contacts is made in which direct protein-protein hydrogen bonds are replaced by protein-water-protein hydrogen bonds as well as a novel hydrogen bond involving the phenolic hydroxyl of the substituted tyrosine.     

Participation of residue F552 in domain III of the protective antigen in the biological activity of anthrax lethal toxin

The protective antigen (PA) component of anthrax toxin translocates the catalytic moieties lethal factor (LF) and edema factor (EF) into the cytosol. The proteolytically activated 63 kDa form of PA (PA63) has the ability to oligomerize and bind LF/EF. PA has four distinct domains performing specialized functions; whereas the function of domains I, II and IV has been well characterized, domain III has no known role in the biological activity of PA. Here we report the role of amino acid residues lining an exposed hydrophobic patch of domain III in the biological activity of PA. The residues Phe552, Phe554, lIe562, Leu566 and lle574 were individually substituted with alanine and the effect was studied. All mutant PA proteins except Phe552Ala were equally active as wild-type PA in exhibiting a toxic phenotype to J774A.1 cells in the presence of LF. Substitution of Ala for Phe552 reduced the ability of PA to intoxicate cells by more than 250-fold. However, Phe552Ala was equally active in receptor binding and susceptibility to trypsin and chymotrypsin as wild-type PA, the activities that have been shown to be essential for the biological activity of PA. This mutated PA protein had a decreased ability to bind LF, oligomerize on cells and to induce release of 86Rb+ from Chinese hamster ovary cells. These results suggest that the residue Phe552 in PA plays an important role in LF binding and oligomerization. Our study provides a basis for further exploration of the biological significance of domain III of PA.     

VP40 octamers are essential for Ebola virus replication

Matrix protein VP40 of Ebola virus is essential for virus assembly and budding. Monomeric VP40 can oligomerize in vitro into RNA binding octamers, and the crystal structure of octameric VP40 has revealed that residues Phe125 and Arg134 are the most important residues for the coordination of a short single-stranded RNA. Here we show that full-length wild-type VP40 octamers bind RNA upon HEK 293 cell expression. While the Phe125-to-Ala mutation resulted in reduced RNA binding, the Arg134-to-Ala mutation completely abolished RNA binding and thus octamer formation. The absence of octamer formation, however, does not affect virus-like particle (VLP) formation, as the VLPs generated from the expression of wild-type VP40 and mutated VP40 in HEK 293 cells showed similar morphology and abundance and no significant difference in size. These results strongly indicate that octameric VP40 is dispensable for VLP formation. The cellular localization of mutant VP40 was different from that of wild-type VP40. While wild-type VP40 was present in small patches predominantly at the plasma membrane, the octamer-negative mutants were found in larger aggregates at the periphery of the cell and in the perinuclear region. We next introduced the Arg134-to-Ala and/or the Phe125-to-Ala mutation into the Ebola virus genome. Recombinant wild-type virus and virus expressing the VP40 Phe125-to-Ala mutation were both rescued. In contrast, no recombinant virus expressing the VP40 Arg134-to-Ala mutation could be recovered. These results suggest that RNA binding of VP40 and therefore octamer formation are essential for the Ebola virus life cycle.     

Thermodynamic consequences of grafting enhanced affinity toward the mutated antigen onto an antibody. The case of anti-lysozyme antibody, HyHEL-10

In order to address the mechanism of enhancement of the affinity of an antibody toward an antigen from a thermodynamic viewpoint, anti-hen lysozyme (HEL) antibody HyHEL-10, which also recognize the mutated antigen turkey lysozyme (TEL) with reduced affinity, was examined. Grafting high affinity toward TEL onto HyHEL-10 was performed by saturation mutagenesis into four residues (Tyr(53), Ser(54), Ser(56), and Tyr(58)) in complementarity-determining region 2 of the heavy chain (CDR-H2) followed by selection with affinity for TEL. Several clones enriched have a Phe residue at site 58. Thermodynamic analyses showed that the clones selected had experienced a greater than 3-fold affinity increase toward TEL in comparison with wild-type Fv, originating from an increase in negative enthalpy change. Substitution of HyHEL-10 HTyr(58) with Phe led to the increase in negative enthalpy change and to almost identical affinity for TEL in comparison with mutants selected, indicating that mutations at other sites decrease the entropy loss despite little contribution to the affinity for TEL. These results suggest that the affinity of an antibody toward the antigen is enhanced by the increase in enthalpy change by some limited mutation, and excess entropy loss due to the mutation is decreased by other energetically neutral mutations.     

The solution structure and dynamics of an Arc repressor mutant reveal premelting conformational changes related to DNA binding.

The solution structure of the hyperstable MYL mutant (R31M/E36Y/R40L) of the Arc repressor of bacteriophage P22 was determined by NMR spectroscopy and compared to that of the wild-type Arc repressor. A backbone rmsd versus the average of 0.37 A was obtained for the well-defined core region. For both Arc-MYL and the wild-type Arc repressor, evidence for a fast equilibrium between a packed ("in") conformation and an extended ("out") conformation of the side chain of Phe 10 was found. In the MYL mutant, the "out" conformation is more highly populated than in the wild-type Arc repressor. The Phe 10 is situated in the DNA-binding beta-sheet of the Arc dimer. While its "in" conformation appears to be the most stable, the "out" conformation is known to be present in the operator-bound form of Arc, where the Phe 10 ring contacts the phosphate backbone [Raumann, B. E., et al. (1994) Nature 367, 754-757]. As well as DNA binding, denaturation by urea and high temperatures induces the functionally active "out" conformation. With a repacking of the hydrophobic core, this characterizes a premelting transition of the Arc repressor. The dynamical properties of the Arc-MYL and the wild-type Arc repressor were further characterized by 15N relaxation and hydrogen-deuterium exchange experiments. The increased main chain mobility at the DNA binding site compared to that of the core of the protein as well as the reorientation of the side chain of Phe 10 is suggested to play an important role in specific DNA binding.     

Functional relevance of aromatic residues in the first transmembrane domain of P2X receptors

The functional relevance of aromatic residues in the upper part of the transmembrane domain-1 of purinergic P2X receptors (P2XRs) was examined. Replacement of the conserved Tyr residue with Ala had a receptor-specific effect: the P2X1R was non-functional, the P2X2R, P2X4R, and P2X3R exhibited enhanced sensitivity to ATP and αβ-meATP accompanied by prolonged decay of current after washout of agonists, and the P2X7R sensitivity for agonists was not affected, though decay of current was delayed. The replacement of the P2X4R-Tyr42 with other amino acids revealed the relevance of an aromatic residue at this position. Mutation of the neighboring Phe and ipsilateral Tyr/Trp residues, but not the contralateral Phe residue, also affected the P2X2R, P2X3R, and P2X4R function. Double mutation of ipsilateral Tyr42 and Trp46 P2X4R residues restored receptor function, whereas the corresponding P2X2R double mutant was not functional. In contrast, mutation of the contralateral Phe48 residue in the P2X4R-Y42A mutant had no effect. These results indicate that aromatic residues in the upper part of TM1 play important roles in the three-dimensional structure of the P2XRs and that they are required not only for ion conductivity but also for specificity of agonist binding and/or channel gating.     

Crystallographic and Cellular Characterisation of Two Mechanisms Stabilising the Native Fold of α1-Antitrypsin: Implications for Disease and Drug Design

The common Z mutant (Glu342Lys) of α₁-antitrypsin results in the formation of polymers that are retained within hepatocytes. This causes liver disease whilst the plasma deficiency of an important proteinase inhibitor predisposes to emphysema. The Thr114Phe and Gly117Phe mutations border a surface cavity identified as a target for rational drug design. These mutations preserve inhibitory activity but reduce the polymerisation of wild-type native α₁-antitrypsin in vitro and increase secretion in a Xenopus oocyte model of disease. To understand these effects, we have crystallised both mutants and solved their structures. The 2.2 Å structure of Thr114Phe α₁-antitrypsin demonstrates that the effects of the mutation are mediated entirely by well-defined partial cavity blockade and allows in silico screening of fragments capable of mimicking the effects of the mutation. The Gly117Phe mutation operates differently, repacking aromatic side chains in the helix F-β-sheet A interface to induce a half-turn downward shift of the adjacent F helix. We have further characterised the effects of these two mutations in combination with the Z mutation in a eukaryotic cell model of disease. Both mutations increase the secretion of Z α₁-antitrypsin in the native conformation, but the double mutants remain more polymerogenic than the wild-type (M) protein. Taken together, these data support different mechanisms by which the Thr114Phe and Gly117Phe mutations stabilise the native fold of α₁-antitrypsin and increase secretion of monomeric protein in cell models of disease.     

Structure of the amino terminus of a gap junction protein

Charged amino acid residues in the amino terminus of gap junction forming proteins (connexins) form part, if not all, of the transjunctional voltage sensor of gap junction channels and play a fundamental role in ion permeation. Results from studies of the voltage dependence of N-terminal mutants predict that residues 1-10 of Group I connexins lie within the channel pore and that the N-terminus forms the channel vestibule by the creation of a turn initiated by the conserved G12 residue. Here we report that intercellular channels containing mutations of G12 in Cx32 to residues that are likely to interfere with flexibility of this locus (G12S, G12Y, and G12V) do not express junctional currents, whereas a connexin containing a proline residue at G12 (Cx32G12P), which is expected to maintain a structure similar to that of the G12 locus, forms nearly wild-type channels. We have solved the structure of an N-terminal peptide of Cx26 (MDWGTLQSILGGVNK) using 1H 2D NMR. The peptide contains two structured domains connected by a flexible hinge (domain-hinge-domain motif) that would allow the placement of the amino terminus within the channel pore. Residues 1-10 adopt a helical conformation and line the channel entrance while residues 12-15 form an open turn. Overall, there is good agreement between the structural and dynamic features of the N-terminal peptide provided by NMR and the functional studies of the voltage dependence of channels formed by wild-type and N-terminal mutations.     

Characterization of the structure and dynamics of amyloidogenic variants of human lysozyme by NMR spectroscopy

The structures and dynamics of the native states of two mutational variants of human lysozyme, I56T and D67H, both associated with non-neuropathic systemic amyloidosis, have been investigated by NMR spectroscopy. The (1)H and (15)N main-chain amide chemical shifts of the I56T variant are very similar to those of the wild-type protein, but those of the D67H variant are greatly altered for 28 residues in the beta-domain. This finding is consistent with the X-ray crystallographic analysis, which shows that the structure of this variant is significantly altered from that of the wild-type protein in this region. The (1)H-(15)N heteronuclear NOE values show that, with the exception of V121, every residue in the wild-type and I56T proteins is located in tightly packed structures characteristic of the native states of most proteins. In contrast, D67H has a region of substantially increased mobility as shown by a dramatic decrease in heteronuclear NOE values of residues near the site of mutation. Despite this unusual flexibility, the D67H variant has no greater propensity to form amyloid fibrils in vivo or in vitro than has I56T. This finding indicates that it is the increased ability of the variants to access partially folded conformations, rather than intrinsic changes in their native state properties, that is the origin of their amyloidogenicity.     

Cumulative Effect of Amino Acid Replacements Results in Enhanced Thermostability of Potato Type L α-Glucan Phosphorylase

The thermostability of potato type L α-glucan phosphorylase (EC 2.4.1.1) was enhanced by random and site-directed mutagenesis. We obtained three single-residue mutations—Phe39→Leu (F39L), Asn135→Ser (N135S), and Thr706→Ile (T706I)—by random mutagenesis. Although the wild-type enzyme was completely inactivated, these mutant enzymes retained their activity even after heat treatment at 60°C for 2 h. Combinations of these mutations were introduced by site-directed mutagenesis. The simultaneous mutation of two (F39L/N135S, F39L/T706I, and N135S/T706I) or three (F39L/N135S/T706I) residues further increased the thermostability of the enzyme, indicating that the effect of the replacement of the residues was cumulative. The triple-mutant enzyme, F39L/N135S/T706I, retained 50% of its original activity after heat treatment at 65°C for 20 min. Further analysis indicated that enzymes with a F39L or T706I mutation were resistant to possible proteolytic degradation.     

Hydrogen-bonding dynamics between adjacent blades in G-protein beta-subunit regulates GIRK channel activation

Functionally critical domains in the betagamma-subunits of the G-protein (Gbetagamma) do not undergo large structural rearrangements upon binding to other proteins. Here we show that a region containing Ser(67) and Asp(323) of Gbetagamma is a critical determinant of G-protein-gated inwardly rectifying K(+) (GIRK) channel activation and undergoes only small structural changes upon mutation of these residues. Using an interactive experimental and computational approach, we show that mutants that form a hydrogen-bond between positions 67 and 323 do not activate a GIRK channel. We also show that in the absence of hydrogen-bonding between these positions, other factors, such as the displacement of the crucial Ggamma residues Pro(60) and Phe(61), can impair Gbetagamma-mediated GIRK channel activation. Our results imply that the dynamic nature of the hydrogen-bonding pattern in the wild-type serves an important functional role that regulates GIRK channel activation by Gbetagamma and that subtle changes in the flexibility of critical domains could have substantial functional consequences. Our results further strengthen the notion that the dynamic regulation of multiple interactions between Gbetagamma and effectors provides for a complex regulatory process in cellular functions.     

Structural and thermodynamic analysis of the packing of two alpha-helices in bacteriophage T4 lysozyme

Packing interactions in bacteriophage T4 lysozyme were explored by determining the structural and thermodynamic effects of substitutions for Ala98 and neighboring residues. Ala98 is buried in the core of T4 lysozyme in the interface between two alpha-helices. The Ala98 to Val (A98V) replacement is a temperature-sensitive lesion that lowers the denaturation temperature of the protein by 15 degrees C (pH 3.0, delta delta G = -4.9 kcal/mol) and causes atoms within the two helices to move apart by up to 0.7 A. Additional structural shifts also occur throughout the C-terminal domain. In an attempt to compensate for the A98V replacement, substitutions were made for Val149 and Thr152, which make contact with residue 98. Site-directed mutagenesis was used to construct the multiple mutants A98V/T152S, A98V/V149C/T152S and the control mutants T152S, V149C and A98V/V149I/T152S. These proteins were crystallized, and their high-resolution X-ray crystal structures were determined. None of the second-site substitutions completely alleviates the destabilization or the structural changes caused by A98V. The changes in stability caused by the different mutations are not additive, reflecting both direct interactions between the sites and structural differences among the mutants. As an example, when Thr152 in wild-type lysozyme is replaced with serine, the protein is destabilized by 2.6 kcal/mol. Except for a small movement of Val94 toward the cavity created by removal of the methyl group, the structure of the T152S mutant is very similar to wild-type T4 lysozyme. In contrast, the same Thr152 to Ser replacement in the A98V background causes almost no change in stability. Although the structure of A98V/T152S remains similar to A98V, the combination of T152S with A98V allows relaxation of some of the strain introduced by the Ala98 to Val replacement. These studies show that removal of methyl groups by mutation can be stabilizing (Val98----Ala), neutral (Thr152----Ser in A98V) or destabilizing (Val149----Cys, Thr152----Ser). Such diverse thermodynamic effects are not accounted for by changes in buried surface area or free energies of transfer of wild-type and mutant side-chains. In general, the changes in protein stability caused by a mutation depend not only on changes in the free energy of transfer associated with the substitution, but also on the structural context within which the mutation occurs and on the ability of the surrounding structure to relax in response to the substitution.(ABSTRACT TRUNCATED AT 400 WORDS)     

The inhibition of degradation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase by sterol regulatory element binding protein cleavage-activating protein requires four phenylalanine residues in span 6 of HMG-CoA reductase transmembrane domain

3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is the rate-limiting enzyme in the cholesterol biosynthetic pathway. This endoplasmic reticulum membrane protein contains a cytosolic catalytic domain and a transmembrane domain with eight membrane spans that are necessary for sterol-accelerated degradation. Competition experiments showed that wild-type transmembrane domains of HMGR and sterol regulatory element binding protein cleavage-activating protein (SCAP) blocked sterol-accelerated degradation of intact HMGR and HMGal, a model protein containing the membrane domain of HMGR linked to Escherichia coli beta-galactosidase. However, mutant transmembrane domains of HMGR and SCAP whose sterol-sensing functions were abolished did not inhibit sterol-accelerated degradation of HMGR and HMGal. In addition, our mutagenesis studies on HMGal indicated that four Phe residues conserved in span 6 of HMGR and the sterol-sensing domains of other sterol-related proteins are required for the regulated degradation of HMGR. These results suggest that HMGR and SCAP compete for binding to a sterol-regulated regulator protein, and this binding may need the four Phe residues.     

Conformational flexibility in the apolipoprotein E amino-terminal domain structure determined from three new crystal forms: implications for lipid binding

An amino-terminal fragment of human apolipoprotein E3 (residues 1-165) has been expressed and crystallized in three different crystal forms under similar crystallization conditions. One crystal form has nearly identical cell dimensions to the previously reported orthorhombic (P2(1)2(1)2(1)) crystal form of the amino-terminal 22 kDa fragment of apolipoprotein E (residues 1-191). A second orthorhombic crystal form (P2(1)2(1)2(1) with cell dimensions differing from the first form) and a trigonal (P3(1)21) crystal form were also characterized. The structures of the first orthorhombic and the trigonal form were determined by seleno-methionine multiwavelength anomalous dispersion, and the structure of the second orthorhombic form was determined by molecular replacement using the structure from the trigonal form as a search model. A combination of modern experimental and computational techniques provided high-quality electron-density maps, which revealed new features of the apolipoprotein E structure, including an unambiguously traced loop connecting helices 2 and 3 in the four-helix bundle and a number of multiconformation side chains. The three crystal forms contain a common intermolecular, antiparallel packing arrangement. The electrostatic complimentarity observed in this antiparallel packing resembles the interaction of apolipoprotein E with the monoclonal antibody 2E8 and the low density lipoprotein receptor. Superposition of the model structures from all three crystal forms reveals flexibility and pronounced kinks in helices near one end of the four-helix bundle. This mobility at one end of the molecule provides new insights into the structural changes in apolipoprotein E that occur with lipid association.     

Alanine-scanning mutagenesis of plasmatocyte spreading peptide identifies critical residues for biological activity

Plasmatocyte spreading peptide (PSP) is a 23-amino acid cytokine that induces a class of insect immune cells called plasmatocytes to spread on foreign surfaces. The structure of PSP consists of a disordered N terminus (residues 1-6) and a well-defined core (residues 7-23) stabilized by a disulfide bridge between Cys(7) and Cys(19), hydrophobic interactions, and a short beta-hairpin. Structural comparisons also indicate that the core region of PSP adopts an epidermal growth factor (EGF)-like fold very similar to the C-terminal subdomain of EGF-like module 5 of thrombomodulin. To identify residues important for plasmatocyte spreading activity, we bioassayed PSP mutants in which amino acids were either replaced with alanine or deleted. Within the well-defined core of PSP, alanine replacement of Cys(7) and Cys(19) (C7.19A) eliminated all activity. Alanine replacement of Arg(13) reduced activity approximately 1000-fold in comparison to wild-type PSP, whereas replacement of the other charged residues (Asp(16), Arg(18), Lys(20)) surrounding Cys(19) diminished activity to a lesser degree. The point mutants Y11A, T14A, T22A, and F23A had activity identical or only slightly reduced to that of wild-type PSP. The mutant PSP-(7-23) lacked the entire unstructured domain of PSP and was found to have no plasmatocyte spreading activity. Surprisingly, E1A and N2A had higher activity than wild-type PSP, but F3A had almost no activity. We thus concluded that the lack of activity for PSP-(7-23) was largely due to the critical importance of Phe(3). To determine whether reductions in activity correlated with alterations in tertiary structure, we compared the C7.19A, R13A, R18A, and F3A mutants to wild-type PSP by NMR spectroscopy. As expected, the simultaneous replacement of Cys(7) and Cys(19) profoundly affected tertiary structure, but the R13A, R18A, and F3A mutants did not differ from wild-type PSP. Collectively, these results indicate that residues in both the unstructured and structured domains of PSP are required for plasmatocyte-spreading activity.     

Crystal and molecular structure of the doubly unsaturated dehydropeptide Ac-delta Phe-Ala-delta Phe-NH-Me.

The dehydropeptide Ac-delta Phe-L-Ala-delta Phe-NH-Me, containing two dehydro-phenylalanine (delta Phe) residues, crystallizes from methanol/water in space group P2(1)2(1)2(1), with a = 12.508 (2), b = 12.746 (1) and c = 15.465 (9). In the crystalline state, the peptide chain assumes a right-handed 3(10)-helical conformation stabilized by two intramolecular hydrogen bonds, between the N-terminal acetyl group and the NH of delta Phe3, and between the CO of delta Phe1 and the NH of the C-terminal methylamide group, respectively. The two consecutive 10-membered rings formed by the hydrogen bonds have torsion angles quite close to the standard values for type III beta-bends. delta Phe1 is located in the (i + 1) position of the first beta-bend, while delta Phe2 is located in the (i + 2) position of the other beta-bend. In the crystal, the molecules are linked head to tail by intermolecular hydrogen bonds to form long helical chains. The axes of the helices are parallel to the c axis, but neighboring helices run in antiparallel directions. This crystal packing is similar to the packing motifs frequently observed in Aib-containing peptides.