Relevant role of Leu265 in helix VI of the angiotensin AT1 receptor in agonist binding and activity

The finding of critical residues for angiotensin II (AII) binding and receptor signalling in helices V and VI led us to assess if, in this region of the receptor, aliphatic side chains might play a role in the agonist-mediated mechanism. Two mutations of the angiotensin AT1 receptor were designed to explore a possible role of a leucine at two positions, Leu265 and Leu268. Thus two mutants, L265D and L268D, were prepared through single substitutions of Leu265, located in the C-terminal region of transmembrane VI (TM-VI), and Leu268, in the adjoining region of the third extracellular loop (EC-3), for an aspartyl residue, and were stably transfected into Chinese hamster ovary (CHO) cells. Ligand-binding experiments and the functional assays determining inositol phosphate (IP) production were performed in these cells expressing these mutants. No significant changes were found in the binding affinity for the ligands, AII, DuP753, and [Sar1Leu8]AII in the mutant L268D. Moreover, the relative potency and the maximum effect on IP production of this mutant were similar to those of the wild-type receptor. In contrast, L265D mutant AT1 receptor, located within the transmembrane domain, markedly decreased binding affinity and ability to stimulate phosphatidylinositol turnover. Our results suggest that the hydrophobic side chain of Leu265, at the C-terminal portion of the AT1's TM-VI, but not Leu268, which belongs to the EC-3 loop, might interact with the AII molecule. On the other side, the aliphatic side chain of Leu265 may be involved in the formation of the ligand binding sites through allosteric effects, thus helping to stabilize the receptor structure around the agonist binding site for full activity.     

Role of amino acid side chains in region 17-31 of parathyroid hormone (PTH) in binding to the PTH receptor

The principal receptor-binding domain (Ser(17)-Val(31)) of parathyroid hormone (PTH) is predicted to form an amphiphilic alpha-helix and to interact primarily with the N-terminal extracellular domain (N domain) of the PTH receptor (PTHR). We explored these hypotheses by introducing a variety of substitutions in region 17-31 of PTH-(1-31) and assessing, via competition assays, their effects on binding to the wild-type PTHR and to PTHR-delNt, which lacks most of the N domain. Substitutions at Arg(20) reduced affinity for the intact PTHR by 200-fold or more, but altered affinity for PTHR-delNt by 4-fold or less. Similar effects were observed for Glu substitutions at Trp(23), Leu(24), and Leu(28), which together form the hydrophobic face of the predicted amphiphilic alpha-helix. Glu substitutions at Arg(25), Lys(26), and Lys(27) (which forms the hydrophilic face of the helix) caused 4-10-fold reductions in affinity for both receptors. Thus, the side chains of Arg(20), together with those composing the hydrophobic face of the ligand's putative amphiphilic alpha-helix, contribute strongly to PTHR-binding affinity by interacting specifically with the N domain of the receptor. The side chains projecting from the opposite helical face contribute weakly to binding affinity by different mechanisms, possibly involving interactions with the extracellular loop/transmembrane domain region of the receptor. The data help define the roles that side chains in the binding domain of PTH play in the PTH-PTHR interaction process and provide new clues for understanding the overall topology of the bimolecular complex.     

Backbone-methylated analogues of the principle receptor binding region of human parathyroid hormone. Evidence for binding to both the N-terminal extracellular domain and extracellular loop region

We have used backbone N-methylations of parathyroid hormone (PTH) to study the role of these NH groups in the C-terminal amphiphilic alpha-helix of PTH (1-31) in binding to and activating the PTH receptor (P1R). The circular dichroism (CD) spectra indicated the structure of the C-terminal alpha-helix was locally disrupted around the methylation site. The CD spectra differences were explained by assuming a helix disruption for four residues on each side of the site of methylation and taking into account the known dependence of CD on the length of an alpha-helix. Binding and adenylyl cyclase-stimulating data showed that outside of the alpha-helix, methylation of residues Asp30 and Val31 had little effect on structure or activities. Within the alpha-helix, disruption of the structure was associated with increased loss of activity, but for specific residues Val21, Leu24, Arg25, and Leu28 there was a dramatic loss of activities, thus suggesting a more direct role of these NH groups in correct P1R binding and activation. Activity analyses with P1R-delNT, a mutant with its long N-terminal region deleted, gave a different pattern of effects and implicated Ser17, Trp23, and Lys26 as important for its PTH activation. These two groups of residues are located on opposite sides of the helix. These results are compatible with the C-terminal helix binding to both the N-terminal segment and also to the looped-out extracellular region. These data thus provide direct evidence for important roles of the C-terminal domain of PTH in determining high affinity binding and activation of the P1R receptor.     

The steady-state kinetics of the enzyme reaction tested by site-directed mutagenesis of hydrophobic residues (Val, Leu, and Cys) in the C-terminal alpha-helix of human adenylate kinase

To elucidate whether the C-terminal region in human adenylate kinase participates in the interaction with the substrate (MgATP(2-) and/or AMP(2-)), hydrophobic residues (Val182, Val186, Cys187, Leu190, and Leu193) were substituted by site-directed mutagenesis and the steady-state kinetics of fifteen mutants were analyzed. A change in the hydrophobic residues in the C-terminal domain affects the affinity for substrates (K(m)), that is, not only for MgATP(2-) but also for AMP(2-), and the catalytic efficiency (k(cat)). The results obtained have led to the following conclusions: (i) Val182 may interact with both MgATP(2-) and AMP(2-) substrates, but to a greater extent with MgATP(2-), and play a role in catalysis. (ii) Val186 appears to play a functional role in catalysis by interacting with both MgATP(2-) and AMP(2-) to nearly the same extent. (iii) Cys187 appears to play a functional role in catalysis. (iv) Leu190 appears to interact with both MgATP(2-) and AMP(2-) substrates but to a greater extent with AMP(2-). (v) Leu193 appears to interact with both MgATP(2-) and AMP(2-) but to a greater extent with AMP(2-). The activity of all mutants decreased due to the change in substrate-affinity. The closer the residue is located to the C-terminal end, the more its mutation affects not only MgATP(2-) but also AMP(2-) substrate binding. The hydrophobic alterations disrupt hydrophobic interactions with substrates and that might destabilize the conformation of the active site. The more C-terminal part of the alpha-helix appears to interact with AMP, as if it has swung out and rotated to cover the adenine moieties. The C-terminal alpha-helix of human adenylate kinase appears to be essential for the interaction with adenine substrates by swinging out during catalysis.     

Importance of each residue within secretin for receptor binding and biological activity

Secretin is a linear 27-residue peptide hormone that stimulates pancreatic and biliary ductular bicarbonate and water secretion by acting at its family B G protein-coupled receptor. While, like other family members, the carboxyl-terminal region of secretin is most important for high affinity binding and its amino-terminal region is most important for receptor selectivity and receptor activation, determinants for these activities are distributed throughout the entire length of this peptide. In this work, we have systematically investigated changing each residue within secretin to alanine and evaluating the impact on receptor binding and biological activity. The residues most critical for receptor binding were His1, Asp3, Gly4, Phe6, Thr7, Ser8, Leu10, Asp15, Leu19, and Leu23. The residues most critical for biological activity included His1, Gly4, Thr7, Ser8, Glu9, Leu10, Leu19, Leu22, and Leu23, with Asp3, Phe6, Ser11, Leu13, Asp15, Leu26, and Val27 also contributing. While the importance of residues in positions analogous to His1, Asp3, Phe6, Thr7, and Leu23 is conserved for several closely related members of this family, Leu19 is uniquely important for secretin. We, therefore, have further studied this residue by molecular modeling and molecular dynamics simulations. Indeed, the molecular dynamics simulations showed that mutation of Leu19 to alanine was destabilizing, with this effect greater than that observed for the analogous position in the other close family members. This could reflect reduced contact with the receptor or an increase in the solvent-accessible surface area of the hydrophobic residues in the carboxyl terminus of secretin as bound to its receptor.     

Inhibition of spontaneous receptor phosphorylation by residues in a putative alpha-helix in the KIT intracellular juxtamembrane region

KIT receptor kinase activity is repressed, prior to stem cell factor binding, by unknown structural constraints. Using site-directed mutagenesis, we examined the role of KIT intracellular juxtamembrane residues Met-552 through Ile-563 in controlling receptor autophosphorylation. Alanine substitution for Tyr-553, Trp-557, Val-559, or Val-560, all sitting along the hydrophobic side of an amphipathic alpha-helix (Tyr-553-Ile-563) predicted by the Chou-Fasman algorithm, resulted in substantially increased spontaneous receptor phosphorylation, revealing inhibitory roles for these residues. Alanine substitution for other residues, most of which are on the hydrophilic side of the helix, caused no or slightly increased basal receptor phosphorylation. Converting Tyr-553 or Trp-557 to phenylalanine generated slight or no elevation, respectively, in basal KIT phosphorylation, indicating that the phenyl ring of Tyr-553 and the hydrophobicity of Trp-557 are critical for the inhibition. Although alanine substitution for Lys-558 had no effect on receptor phosphorylation, its substitution with proline produced high spontaneous receptor phosphorylation, suggesting that the predicted alpha-helical conformation is involved in the inhibition. A synthetic peptide comprising Tyr-553 through Ile-563 showed circular dichroism spectra characteristic of alpha-helix, supporting the structural prediction. Thus, the KIT intracellular juxtamembrane region contains important residues which, in a putative alpha-helical conformation, exert inhibitory control on the kinase activity of ligand-unoccupied receptor.     

Preferred conformations of RGDX tetrapeptides to inhibit the binding of fibrinogen to platelets

The conformational study on Arg-Gly-Asp (RGD)-containing tetrapeptides in the unhydrated and hydrated states has been carried out using the force field ECEPP/3 and the hydration shell model. The tetrapeptides studied here are H-RGDX-OH (X = Trp, Tyr, Phe, Leu, Val, Cys, Gln, and Ser), which show the inhibitory activity for binding of fibrinogen to platelets in the order of RGDW approximately equal to RGDY approximately equal to RGDF approximately equal to RGDL > RGDV > or = RGDC > or = RGDQ > or = RGDS. The backbone conformations with two C(7) backbone-to-backbone hydrogen bonds between Asp and Arg residues and between Xaa and Gly residues are in common most probable for the RGD sequence of RGDX tetrapeptides in the hydrated state. The dominant beta-turns for RGDX are found to be the types V' and IV at Gly-Asp and Asp-Xaa sequences, respectively, which are quite similar to the types II' and I (or II), respectively. However, it cannot be ruled out that the extended conformations are also remarkably feasible for RGDX tetrapeptides in water by peering the distributions of backbone conformations. These calculated results are consistent with the experimental results on RGD-containing proteins and conformationally constrained RGD-containing peptides. The reason why the RGDX becomes more potent as the side chain of the X residue is more hydrophobic may be ascribed to that the more hydrophobic is the residue X, the more populated are beta-turn structures for the Gly-Asp sequence. The hydrophobic side chain of X residue exposed to water is likely to interact with the hydrophobic region of receptor easily.     

Mutational analysis of residues in the helical region of the class IIa bacteriocin pediocin PA-1

A 15-mer fragment that is derived from the helical region in the C-terminal half of pediocin PA-1 inhibited the activity of pediocin PA-1. Of 13 other pediocin-like (hybrid) bacteriocins, only the hybrid bacteriocin Sak/Ped was markedly inhibited by the 15-mer fragment. Sak/Ped was the only one of these bacteriocins that had a sequence (in the C-terminal helix-containing half) identical to that of the 15-mer fragment, indicating that the fragment inhibits pediocin-like bacteriocins in a sequence-dependent manner. By replacing (one at a time) all 15 residues in the fragment with Ala or Leu, five residues (K1, A2, T4, N8, and A15) were identified as being especially important for the inhibitory action of the fragment. The results suggest that the corresponding residues (K20, A21, T23, N27, and A34, respectively) in pediocin PA-1 might be involved in interactions between pediocin PA-1 and its receptor. To characterize the environment surrounding these five residues when pediocin PA-1 interacts with target cells, these residues were replaced (one at a time) with a hydrophobic large (Leu) residue, a hydrophilic charged (Asp or Arg) residue, and a small (Ala or Gly) residue. The results revealed that residues A21 and A34 are in a spatially constrained environment, since the replacement with a small (Gly) residue was the only substitution that did not markedly reduce the bacteriocin activity. The positive charge in K20 and the polar amide group in N27 appeared to interact with electronegative groups, since the replacement of these two residues with a positive (Arg) residue was well tolerated, while replacement with a negative (Asp) residue was detrimental to the bacteriocin activity. K20 was in a less constrained environment than N27, since the replacement of K20 with a large hydrophobic (Leu) residue was tolerated fairly well and to a greater extent than N27. T23 seemed to be in an environment that was not restricted with respect to size, polarity, and charge, since replacements with large (Leu) and small (Ala) hydrophobic residues and a hydrophilic negative (Asp) residue were tolerated fairly well (2- to 6-fold reduction in activity). Moreover, the replacement of T23 with a large positive (Arg) residue resulted in wild-type or better-than-wild-type activity.     

Consequences of single-site mutations in the intestinal fatty acid binding protein

The intestinal fatty acid binding protein (IFABP) is a small (15 kDa) protein consisting mostly of 10 antiparallel beta-strands (A-J) and a small helical region that serves as a portal for the ligand. Two beta-sheet structures (strands A-E and F-J) surround a cavity into which the ligand binds. In this work, we investigated how changes in the side chains of specific residues are propagated through the structure. To determine what these changes were and how they relate to changes in stability, (15)N chemical shift perturbations were measured and compared to those of the wild-type protein. Seven mutations, five of which change either valine or leucine to glycine, have been examined. All these mutants were less stable than wild-type IFABP, suggesting some structural changes. For five of the mutants, the data suggest that destabilization of a small region of the protein propagates throughout the structure, resulting in an overall decrease in stability. In two (Leu38Gly and Leu89Gly), the loss of cooperativity in the equilibrium denaturation curves suggests that the destabilization of one region may not be transmitted to other regions in a cooperative manner. It is shown that the effect of mutating hydrophobic residues is much greater than that observed upon mutation of a solvent-exposed polar residue.     

Role of the hinge region and the tryptophan residue in the synthetic antimicrobial peptides, cecropin A(1-8)-magainin 2(1-12) and its analogues, on their antibiotic activities and structures

A 20-residue hybrid peptide CA(1-8)-MA(1-12) (CA-MA), incorporating residues 1-8 of cecropin A (CA) and residues 1-12 of magainin 2 (MA), has potent antimicrobial activity without toxicity against human erythrocytes. To investigate the effects of the Gly-Ile-Gly hinge sequence of CA-MA on the antibacterial and antitumor activities, two analogues in which the Gly-Ile-Gly sequence of CA-MA is either deleted (P1) or substituted with Pro (P2) were synthesized. The role of the tryptophan residue at position 2 of CA-MA on its antibiotic activity was also investigated using two analogues, in which the Trp2 residue of CA-MA is replaced with either Ala (P3) or Leu (P4). The tertiary structures of CA-MA, P2, and P4 in DPC micelles, as determined by NMR spectroscopy, have a short amphiphilic helix in the N-terminus and about three turns of alpha-helix in the C-terminus, with the flexible hinge region between them. The P1 analogue has an alpha-helix from Leu4 to Ala14 without any hinge structure. P1 has significantly decreased lytic activities against bacterial and tumor cells and PC/PS vesicles (3:1, w/w), and reduced pore-forming activity on lipid bilayers, while P2 retained effective lytic activities and pore-forming activity. The N-terminal region of P3 has a flexible structure without any specific secondary structure. The P3 modification caused a drastic decrease in the antibiotic activities, whereas P4, with the hydrophobic Leu side chain at position 2, retained its activities. On the basis of the tertiary structures, antibiotic activities, vesicle-disrupting activities, and pore-forming activities, the structure-function relationships can be summarized as follows. The partial insertion of the Trp2 of CA-MA into the membrane, as well as the electrostatic interactions between the positively charged Lys residues at the N-terminus of the CA-MA and the anionic phospholipid headgroups, leads to the primary binding to the cell membrane. Then, the flexibility or bending potential induced by the Gly-Ile-Gly hinge sequence or the Pro residue in the central part of the peptides may allow the alpha-helix in the C-terminus to span the lipid bilayer. These structural features are crucial for the potent antibiotic activities of CA-MA.     

Identification of a receptor binding site in the carboxyl terminus of human interleukin-6.

To identify a receptor binding site of human interleukin-6 (IL-6), we created a library of IL-6 variants with single amino acid substitutions in the last 15 residues (171-185) in the COOH terminus of IL-6. Twenty-seven IL-6 variants were tested for biological activity on a human hepatoma and a mouse hybridoma cell line. Most variants were additionally tested in a receptor binding assay using a human myeloma cell line. Several single amino acid substitutions in the COOH terminus of IL-6 were found to decrease biological activity significantly. This is especially seen in variants with amino acid substitutions that alter the postulated amphipathical alpha-helix structure between residues 178 and 183. The two highly conserved Arg residues at positions 180 and 183 seem to play a very important role in biological activity. The loss of biological activity in all inactive variants is completely paralleled by a decrease of IL-6 receptor binding, as determined by competition binding experiments. One mutant (Leu171) displayed a higher activity on human cells and a higher binding affinity to the receptor and can be considered an IL-6 agonist. It is concluded that the amphipathical alpha-helix structure in the COOH terminus of IL-6 is critical for ligand receptor interaction. Furthermore, the region between residues Ser178 and Arg183 (Ser-Leu-Arg-Ala-X-Arg) is identified as a receptor binding site in the COOH terminus of human IL-6.     

Analysis of antibody A6 binding to the extracellular interferon gamma receptor alpha-chain by alanine-scanning mutagenesis and random mutagenesis with phage display

The monoclonal antibody A6 binds a conformational epitope comprising mainly the CC' surface loop on the N-terminal fibronectin type-III domain of the extracellular interferon gamma receptor (IFNgammaR). The crystal structure of an A6 Fab-IFNgammaR complex revealed an interface rich in the aromatic side chains of Trp, Tyr, and His residues. These aromatic side chains appear to interact with both polar and hydrophobic groups at the interface, a property which, in general, may be advantageous for ligand binding. To analyze these interactions in more detail, the affinities of 19 A6 alanine-scanning mutants for the IFNgammaR have been measured, using engineered A6 single chain variable region fragments, and a surface plasmon resonance biosensor. Energetically important side chains (DeltaG(mutant) - DeltaG(wt) > 2.4 kcal/mol), that form distinct hot spots in the binding interface, have been identified on both proteins. These include V(L)W92 in A6, whose benzenoid ring appears well situated for a pi-cation (or pi-amine) interaction with the side chain of receptor residue K47 and simultaneously for T-stacking onto the indole ring of W82 in the receptor. At another site, energetically important residues V(H)W52 and V(H)W53, as well as V(H)D54 and V(H)D56, surround the aliphatic side chain of the hot receptor residue K52. Taken together, the results show that side chains distributed across the interface, including many aromatic ones, make key energetic contributions to binding. In addition, the receptor CC' loop has been subjected to random mutagenesis, and receptor mutants with high affinity for A6 have been selected by phage display. Residues previously identified as important for receptor binding to A6 were conserved in the clones isolated. Some mutants, however, showed a much improved affinity for A6, due to changes at Glu55, a residue that appeared to be energetically unimportant for binding the antibody by alanine-scanning mutagenesis. An E55P receptor mutant bound A6 with a 600-fold increase in affinity (K(D) approximately 20 pM), which is one of the largest improvements in affinity from a single point mutation reported so far at any protein-protein interface.     

Characterization of the calcyclin (S100A6) binding site of annexin XI-A by site-directed mutagenesis

Residues in annexin XI-A essential for binding of calcyclin (S100A6) were examined by site-directed mutagenesis. GST fusion proteins with the calcyclin binding site of annexin XI-A, GST-AXI 34-62 and GST-AXI 49-77 bound to calcyclin-Sepharose Ca2+-dependently. The mutants GST-AXI L52E, M55E, A56E and M59E lost the binding ability, whereas GST-AXI A57E retained the ability. These results demonstrate that the hydrophobic residues L52, M55, A56 and M59 on one side surface of the alpha-helix are critical for the binding. Assays with GST fusion proteins and synthesized peptides corresponding to the calcyclin binding site indicated that other regions around the calcyclin binding site are important to stabilize the conformation.     

The specificity of carboxypeptidase Y may be altered by changing the hydrophobicity of the S'1 binding pocket.

The S'1 binding pocket of carboxypeptidase Y is hydrophobic, spacious, and open to solvent, and the enzyme exhibits a preference for hydrophobic P'1 amino acid residues. Leu272 and Ser297, situated at the rim of the pocket, and Leu267, slightly further away, have been substituted by site-directed mutagenesis. The mutant enzymes have been characterized kinetically with respect to their P'1 substrate preferences using the substrate series FA-Ala-Xaa-OH (Xaa = Leu, Glu, Lys, or Arg) and FA-Phe-Xaa-OH (Xaa = Ala, Val, or Leu). The results reveal that hydrophobic P'1 residues bind in the vicinity of residue 272 while positively charged P'1 residues interact with Ser297. Introduction of Asp or Glu at position 267 greatly reduced the activity toward hydrophobic P'1 residues (Leu) and increased the activity two- to three-fold for the hydrolysis of substrates with Lys or Arg in P'1. Negatively charged substituents at position 272 reduced the activity toward hydrophobic P'1 residues even more, but without increasing the activity toward positively charged P'1 residues. The mutant enzyme L267D + L272D was found to have a preference for substrates with C-terminal basic amino acid residues. The opposite situation, where the positively charged Lys or Arg were introduced at one of the positions 267, 272, or 297, did not increase the rather low activity toward substrates with Glu in the P'1 position but greatly reduced the activity toward substrates with C-terminal Lys or Arg due to electrostatic repulsion. The characterized mutant enzymes exhibit various specificities, which may be useful in C-terminal amino acid sequence determinations.     

Identification of amino acids involved in protein structural uniqueness: implication for de novo protein design

Structural uniqueness is characteristic of native proteins and is essential to express their biological functions. The major factors that bring about the uniqueness are specific interactions between hydrophobic residues and their unique packing in the protein core. To find the origin of the uniqueness in their amino acid sequences, we analyzed the distribution of the side chain rotational isomers (rotamers) of hydrophobic amino acids in protein tertiary structures and derived deltaS(contact), the conformational-entropy changes of side chains by residue-residue contacts in each secondary structure. The deltaS(contact) values indicate distinct tendencies of the residue pairs to restrict side chain conformation by inter-residue contacts. Of the hydrophobic residues in alpha-helices, aliphatic residues (Leu, Val, Ile) strongly restrict the side chain conformations of each other. In beta-sheets, Met is most strongly restricted by contact with Ile, whereas Leu, Val and Ile are less affected by other residues in contact than those in alpha-helices. In designed and native protein variants, deltaS(contact) was found to correlate with the folding-unfolding cooperativity. Thus, it can be used as a specificity parameter for designing artificial proteins with a unique structure.     

Stable proline box motif at the N-terminal end of alpha-helices.

We describe a novel N-terminal alpha-helix local motif that involves three hydrophobic residues and a Pro residue (Pro-box motif). Database analysis shows that when Pro is the N-cap of an alpha-helix the distribution of amino acids in adjacent positions changes dramatically with respect to the average distribution in an alpha-helix, but not when Pro is at position N1. N-cap Pro residues are usually associated to Ile and Leu, at position N', Val at position N3 and a hydrophobic residue (h) at position N4. The side chain of the N-cap Pro packs against Val, while the hydrophobic residues at positions N' and N4 make favorable interactions. To analyze the role of this putative motif (sequence fingerprint hPXXhh), we have synthesized a series of peptides and analyzed them by circular dichroism (CD) and NMR. We find that this motif is formed in peptides, and that the accompanying hydrophobic interactions contribute up to 1.2 kcal/mol to helix stability. The fact that some of the residues in this fingerprint are not good N-cap and helix formers results in a small overall stabilization of the alpha-helix with respect to other peptides having Gly as the N-cap and Ala at N3 and N4. This suggests that the Pro-box motif will not specially contribute to protein stability but to the specificity of its fold. In fact, 80% of the sequences that contain the fingerprint sequence in the protein database are adopting the described structural motif, and in none of them is the helix extended to place Pro at the more favorable N1 position.     

Identification of key residues in the amino-terminal third of human interleukin-1 alpha

Two mutational approaches were used to perform a thorough structure-function analysis of the first 53 residues of the 159-residue cytokine human interleukin-1 alpha (hIL-1 alpha). In this study, a total of 26 deletions, 97 multiple amino acid substitutions, and 46 single amino acid substitutions were examined. A synthetic hIL-1 alpha gene with many unique restriction sites was constructed to facilitate the molecular manipulations that were performed. The mutational methods employed include: Bal-31 exonuclease-generated deletions at unique restriction sites and combinatorial cassette mutagenesis via segment replacement with synthetic DNA. The mutant hIL-1 alpha proteins were expressed at high levels in Escherichia coli and were assayed for biological activity in a mouse T cell proliferation assay. We observed that the activity of hIL-1 alpha was extraordinarily sensitive to deletion mutations. Most internal deletions of as few as 1 or 2 residues substantially reduced biological activity. Combinatorial cassette mutagenesis on residues 13-53 of hIL-1 alpha identified 15 important residue positions. Of these, 8 displayed strong preferences for residues with hydrophilic side chains, and the remainder preferred hydrophobic side chains. We found that functional hIL-1 alpha had an absolute requirement for a basic residue (Arg, Lys, or His) at either position 15 or 16, and that Leu was preferred at position 40.     

Hydrophobic residues within the predicted N-terminal amphiphilic alpha-helix of a plant mitochondrial targeting presequence play a major role in in vivo import

A deletion and mutagenesis study was performed on the mitochondrial presequence of the beta-subunit of the F(1)-ATP synthase from Nicotiana plumbaginifolia linked to the green fluorescent protein (GFP). The various constructs were tested in vivo by transient expression in tobacco protoplasts. GFP distribution in transformed cells was analysed in situ by confocal microscopy, and in vitro in subcellular fractions by Western blotting. Despite its being highly conserved in different species, deletion of the C-terminal region (residues 48-54) of the presequence did not affect mitochondrial import. Deletion of the conserved residues 40-47 and the less conserved intermediate region (residues 18-39) resulted in 60% reduction in GFP import, whereas mutation of conserved residues within these regions had little effect. Further shortening of the presequence progressively reduced import, with the construct retaining the predicted N-terminal amphiphilic alpha-helix (residues 1-12) being unable to mediate mitochondrial import. However, point mutation showed that this last region plays an important role through its basic residues and amphiphilicity, but also through its hydrophobic residues. Replacing Arg4 and Arg5 by alanine residues and shifting the Arg5 and Leu6 (in order to disturb amphiphilicity) resulted in reduction of the presequence import efficiency. The most dramatic effects were seen with single or double mutations of the four Leu residues (positions 5, 6, 10 and 11), which resulted in marked reduction or abolition of GFP import, respectively. We conclude that the N-terminal helical structure of the presequence is necessary but not sufficient for efficient mitochondrial import, and that its hydrophobic residues play an essential role in in vivo mitochondrial targeting.     

Solution structure and biological activity of recombinant salmon calcitonin S-sulfonated analog

Salmon calcitonin S-sulfonated analog (abbreviated as [S-SO(3)(-)]rsCT) was prepared by introducing two sulfonic groups into the side chains of Cys1 and Cys7 of recombinant salmon calcitonin. The hypocalcemic potency of this open-chain analog is 5500IU/mg, which is about 30% higher than that (4500IU/mg) of the wild type. The solution conformation of [S-SO(3)(-)]rsCT was studied in aqueous trifluoroethanol solution by CD, 2D-NMR spectroscopy, and distance geometry calculations. In the mixture of 60% TFE and 40% water, the peptide assumes an amphipathic alpha-helix in the region of residues 4-22, which is one turn longer than that of the native sCT. The structural feature analysis of the peptide revealed the presence of hydrophobic surface composed of five hydrophobic side chains of residues Leu4, Leu9, Leu12, Leu16, and Leu19, and a network of salt-bridges that consisted of a tetrad of oppositely charged side chains (Cys7-SO(3)(-)-Lys11(+)-Glu15(-)-Lys18(+)). The multiple salt bridges resulted in the stabilization of the longer amphipathic alpha-helix. Meanwhile, the higher hypocalcemic potency of the peptide could be attributed to the array of hydrophobic side chains of five leucine residues of the amphipathic alpha-helix.     

Hydrophobic side-chain size is a determinant of the three-dimensional structure of the p53 oligomerization domain.

The p53 tumor suppressor oligomerization domain, a dimer of two primary dimers, is an independently folding domain whose subunits consist of a beta-strand, a tight turn and an alpha-helix. To evaluate the effect of hydrophobic side-chains on three-dimensional structure, we substituted residues Phe341 and Leu344 in the alpha-helix with other hydrophobic amino acids. Substitutions that resulted in residue 341 having a smaller side-chain than residue 344 switched the stoichiometry of the domain from tetrameric to dimeric. The three-dimensional structure of one such dimer was determined by multidimensional NMR spectroscopy. When compared with the primary dimer of the wild-type p53 oligomerization domain, the mutant dimer showed a switch in alpha-helical packing from anti-parallel to parallel and rotation of the alpha-helices relative to the beta-strands. Hydrophobic side-chain size is therefore an important determinant of a protein fold.