An information theory model of hydrophobic interactions.

A molecular model of poorly understood hydrophobic effects is heuristically developed using the methods of information theory. Because primitive hydrophobic effects can be tied to the probability of observing a molecular-sized cavity in the solvent, the probability distribution of the number of solvent centers in a cavity volume is modeled on the basis of the two moments available from the density and radial distribution of oxygen atoms in liquid water. The modeled distribution then yields the probability that no solvent centers are found in the cavity volume. This model is shown to account quantitatively for the central hydrophobic phenomena of cavity formation and association of inert gas solutes. The connection of information theory to statistical thermodynamics provides a basis for clarification of hydrophobic effects. The simplicity and flexibility of the approach suggest that it should permit applications to conformational equilibria of nonpolar solutes and hydrophobic residues in biopolymers.

Role of hydrophobic interactions and desolvation in determining the structural properties of a model alpha beta peptide.

Model AB, a 20-amino acid peptide that was designed to adopt an alpha beta tertiary structure stabilized by hydrophobic interactions between residues in adjacent helical and extended segments, exhibited large pKa shifts of several ionizable groups and slow hydrogen/deuterium exchange rates of nearly all the peptide amide groups [Butcher, D. J., Bruch, M. D. & Moe, G. T. (1995) Biopolymers 36, 109-120]. These properties, which depend on structure and hydration, are commonly observed in larger proteins but are quite unusual for small peptides. To identify which of several possible features of the peptide design are most important in determining these properties, several closely related analogs of Model AB were characterized by CD and NMR spectroscopy. The results show that hydrophobic interactions between adjacent helical and extended segments are structure-determining and have the additional effect of altering water-peptide interactions over much of the peptide surface. These results may have important implications for understanding mechanisms of protein folding and for the design of independently folding peptides.

Hydrophobic sequence minimization of the α-lactalbumin molten globule

The molten globule, a widespread protein-folding intermediate, can attain a native-like backbone topology, even in the apparent absence of rigid side-chain packing. Nonetheless, mutagenesis studies suggest that molten globules are stabilized by some degree of side-chain packing among specific hydrophobic residues. Here we investigate the importance of hydrophobic side-chain diversity in determining the overall fold of the α-lactalbumin molten globule. We have replaced all of the hydrophobic amino acids in the sequence of the helical domain with a representative amino acid, leucine. Remarkably, the minimized molecule forms a molten globule that retains many structural features characteristic of a native α-lactalbumin fold. Thus, nonspecific hydrophobic interactions may be sufficient to determine the global fold of a protein.

Barriers to protein folding: formation of buried polar interactions is a slow step in acquisition of structure.

In the MYL mutant of the Arc repressor dimer, sets of partially buried salt-bridge and hydrogen-bond interactions mediated by Arg-31, Glu-36, and Arg-40 in each subunit are replaced by hydrophobic interactions between Met-31, Tyr-36, and Leu-40. The MYL refolding/dimerization reaction differs from that of wild type in being 10- to 1250-fold faster, having an earlier transition state, and depending upon viscosity but not ionic strength. Formation of the wild-type salt bridges in a hydrophobic environment clearly imposes a kinetic barrier to folding, which can be lowered by high salt concentrations. The changes in the position of the transition state and viscosity dependence can be explained if denatured monomers interact to form a partially folded dimeric intermediate, which then continues folding to form the native dimer. The second step is postulated to be rate limiting for wild type. Replacing the salt bridge with hydrophobic interactions lowers this barrier for MYL. This makes the first kinetic barrier rate limiting for MYL refolding and creates a downhill free-energy landscape in which most molecules which reach the intermediate state continue to form native dimers.

Surface hydrophobic residues of multiubiquitin chains essential for proteolytic targeting.

Ubiquitin conjugation is a signal for degradation of eukaryotic proteins by the 26S protease. Conjugation of a homopolymeric multiubiquitin chain to a substrate lysine residue results in 10-fold faster degradation than does conjugation of monoubiquitin, but the molecular basis of enhanced targeting by chains is unknown. We show that ubiquitin residues L8, I44, and V70 are critical for targeting. Mutation of pairs of these residues to alanine had little effect on attachment of ubiquitin to substrates but severely inhibited degradation of the resulting conjugates. The same mutations blocked the binding of chains to a specific subunit (S5a) of the regulatory complex of the 26S protease. The side chains implicated in this binding--L8, I44, and V70--form repeating patches on the chain surface. Thus, hydrophobic interactions between these patches and S5a apparently contribute to enhanced proteolytic targeting by multiubiquitin chains.

The putative actin-binding role of hydrophobic residues Trp546 and Phe547 in chicken gizzard heavy meromyosin.

In the course of myosin-catalyzed ATP hydrolysis, certain amino acid residues in myosin interact with counterparts in actin to produce the relational changes that underlie muscle contraction; some of these interactions are ionic, but the stronger interactions are hydrophobic. In an effort to identify myosin residues participating in hydrophobic interactions, myosin (from smooth muscle) fragments with mutations at suspected sites were engineered and compared with wild-type fragments. It was found that the ATPase of doubly mutated (Trp546Ser and Phe547His) fragments was minimally activated by actin and did not decorate actin well to form the regular arrowhead pattern characteristic of myosin binding to actin filaments. Thus, we suggest that Trp546 and Phe547 are important participants in the hydrophobic actin-myosin interaction.

A peptide interaction in the major groove of RNA resembles protein interactions in the minor groove of DNA.

A 17-amino acid arginine-rich peptide from the bovine immunodeficiency virus Tat protein has been shown to bind with high affinity and specificity to bovine immunodeficiency virus transactivation response element (TAR) RNA, making contacts in the RNA major groove near a bulge. We show that, as in other peptide-RNA complexes, arginine and threonine side chains make important contributions to binding but, unexpectedly, that one isoleucine and three glycine residues also are critical. The isoleucine side chain may intercalate into a hydrophobic pocket in the RNA. Glycine residues may allow the peptide to bind deeply within the RNA major groove and may help determine the conformation of the peptide. Similar features have been observed in protein-DNA and drug-DNA complexes in the DNA minor groove, including hydrophobic interactions and binding deep within the groove, suggesting that the major groove of RNA and minor groove of DNA may share some common recognition features.

Structural basis for chemical inhibition of human blood coagulation factor Xa

Factor Xa, the converting enzyme of prothrombin to thrombin, has emerged as an alternative (to thrombin) target for drug discovery for thromboembolic diseases. An inhibitor has been synthesized and the crystal structure of the complex between Des[1–44] factor Xa and the inhibitor has been determined by crystallographic methods in two different crystal forms to 2.3- and 2.4-Å resolution. The racemic mixture of inhibitor FX-2212, (2RS)-(3′-amidino-3-biphenylyl)-5-(4-pyridylamino)pentanoic acid, inhibits factor Xa activity by 50% at 272 nM in vitro. The S-isomer of FX-2212 (FX-2212a) was found to bind to the active site of factor Xa in both crystal forms. The biphenylamidine of FX-2212a occupies the S1-pocket, and the pyridine ring makes hydrophobic interactions with the factor Xa aryl-binding site. Several water molecules meditate inhibitor binding to residues in the active site. In contrast to the earlier crystal structures of factor Xa, such as those of apo-Des[1–45] factor Xa and Des[1–44] factor Xa in complex with a naphthyl inhibitor DX-9065a, two epidermal growth factor-like domains of factor Xa are well ordered in both our crystal forms as well as the region between the two domains, which recently was found to be the binding site of the effector cell protease receptor-1. This structure provides a basis for designing next generation inhibitors of factor Xa.

A model for the structure of the P domains in the subtilisin-like prohormone convertases

The proprotein convertases are a family of at least seven calcium-dependent endoproteases that process a wide variety of precursor proteins in the secretory pathway. All members of this family possess an N-terminal proregion, a subtilisin-like catalytic module, and an additional downstream well-conserved region of ≈150 amino acid residues, the P domain, which is not found in any other subtilase. The pro and catalytic domains cannot be expressed in the absence of the P domains; their thermodynamic instability may be attributable to the presence of large numbers of negatively charged Glu and Asp side chains in the substrate binding region for recognition of multibasic residue cleavage sites. Based on secondary structure predictions, we here propose that the P domains consist of 8-stranded β-barrels with well-organized inner hydrophobic cores, and therefore are independently folded components of the proprotein convertases. We hypothesize further that the P domains are integrated through strong hydrophobic interactions with the catalytic domains, conferring structural stability and regulating the properties and activity of the convertases. A molecular model of these interdomain interactions is proposed in this report.

Inhibiting transthyretin conformational changes that lead to amyloid fibril formation

Insoluble protein fibrils resulting from the self-assembly of a conformational intermediate are implicated as the causative agent in several severe human amyloid diseases, including Alzheimer’s disease, familial amyloid polyneuropathy, and senile systemic amyloidosis. The latter two diseases are associated with transthyretin (TTR) amyloid fibrils, which appear to form in the acidic partial denaturing environment of the lysosome. Here we demonstrate that flufenamic acid (Flu) inhibits the conformational changes of TTR associated with amyloid fibril formation. The crystal structure of TTR complexed with Flu demonstrates that Flu mediates intersubunit hydrophobic interactions and intersubunit hydrogen bonds that stabilize the normal tetrameric fold of TTR. A small-molecule inhibitor that stabilizes the normal conformation of a protein is desirable as a possible approach to treat amyloid diseases. Molecules such as Flu also provide the means to rigorously test the amyloid hypothesis, i.e., the apparent causative role of amyloid fibrils in amyloid disease.

Scanning mutagenesis of the putative transmembrane segments of Kir2.1, an inward rectifier potassium channel

Structural models of inward rectifier K+ channels incorporate four identical or homologous subunits, each of which has two hydrophobic segments (M1 and M2) which are predicted to span the membrane as α helices. Since hydrophobic interactions between proteins and membrane lipids are thought to be generally of a nonspecific nature, we attempted to identify lipid-contacting residues in Kir2.1 as those which tolerate mutation to tryptophan, which has a large hydrophobic side chain. Tolerated mutations were defined as those which produced measurable inwardly rectifying currents in Xenopus oocytes. To distinguish between water-accessible positions and positions adjacent to membrane lipids or within the protein interior we also mutated residues in M1 and M2 individually to aspartate, since an amino acid with a charged side chain should not be tolerated at lipid-facing or interior positions, due to the energy cost of burying a charge in a hydrophobic environment. Surprisingly, 17 out of 20 and 17 out of 22 non-tryptophan residues in M1 and M2, respectively, tolerated being mutated to tryptophan. Moreover, aspartate was tolerated at 15 out of 22 and 15 out of 21 non-aspartate M1 and M2 positions respectively. Periodicity in the pattern of tolerated vs. nontolerated mutations consistent with α helices or β strands did not emerge convincingly from these data. We consider the possibility that parts of M1 and M2 may be in contact with water.

A β-hairpin structure in a 13-mer peptide that binds α-bungarotoxin with high affinity and neutralizes its toxicity

Snake-venom α-bungarotoxin is a member of the α-neurotoxin family that binds with very high affinity to the nicotinic acetylcholine receptor (AChR) at the neuromuscular junction. The structure of the complex between α-bungarotoxin and a 13-mer peptide (WRYYESSLEPYPD) that binds the toxin with high affinity, thus inhibiting its interactions with AChR with an IC50 of 2 nM, has been solved by 1H-NMR spectroscopy. The bound peptide folds into a β-hairpin structure created by two antiparallel β-strands, which combine with the already existing triple-stranded β-sheet of the toxin to form a five-stranded intermolecular, antiparallel β-sheet. Peptide residues Y3P, E5P, and L8P have the highest intermolecular contact area, indicating their importance in the binding of α-bungarotoxin; W1P, R2P, and Y4P also contribute significantly to the binding. A large number of characteristic hydrogen bonds and electrostatic and hydrophobic interactions are observed in the complex. The high-affinity peptide exhibits inhibitory potency that is better than any known peptide derived from AChR, and is equal to that of the whole α-subunit of AChR. The high degree of sequence similarity between the peptide and various types of AChRs implies that the binding mode found within the complex might possibly mimic the receptor binding to the toxin. The design of the high-affinity peptide was based on our previous findings: (i) the detection of a lead peptide (MRYYESSLKSYPD) that binds α-bungarotoxin, using a phage-display peptide library, (ii) the information about the three-dimensional structure of α-bungarotoxin/lead-peptide complex, and (iii) the amino acid sequence analysis of different AChRs.

The C-terminal region of human angiogenin has a dual role in enzymatic activity.

The ribonucleolytic activity of angiogenin (Ang) is essential to Ang's capacity to induce blood vessel formation. Previous x-ray diffraction and mutagenesis results have shown that the active site of the human protein is obstructed by Gln-117 and imply that the C-terminal region of Ang must undergo a conformational rearrangement to allow substrate binding and catalysis. As a first step toward structural characterization of this conformational change, additional site-directed mutagenesis and kinetic analysis have been used to examine the intramolecular interactions that stabilize the inactive conformation of the protein. Two residues of this region, Ile-119 and Phe-120, are found to make hydrophobic interactions with the remainder of the protein and thereby help to keep Gln-117 in its obstructive position. Furthermore, the suppression of activity by the intramolecular interactions of Ile-119 and Phe-120 is counterbalanced by an effect of the adjacent residues, Arg-121, Arg-122, and Pro-123 which do not appear to form contacts with the rest of the protein structure. They contribute to enzymatic activity, probably by constituting a peripheral subsite for binding polymeric substrates. The results reveal the nature of the conformational change in human Ang and assign a key role to the C-terminal region both in this process and, presumably, in the regulation of human Ang function.

High-affinity binding of Escherichia coli SecB to the signal sequence region of a presecretory protein.

The Escherichia coli cytosolic homotetrameric protein SecB is known to be involved in protein export across the plasma membrane. A currently prevalent view holds that SecB functions exclusively as a chaperone interacting nonspecifically with unfolded proteins, not necessarily exported proteins, whereas a contrary view holds that SecB functions primarily as a specific signal-recognition factor--i.e., in binding to the signal sequence region of exported proteins. To experimentally resolve these differences we assayed for binding between chemically pure SecB and chemically pure precursor (p) form (containing a signal sequence) and mature (m) form (lacking a signal sequence) of a model secretory protein (maltose binding protein, MBP) that was C-terminally truncated. Because of the C-terminal truncation, neither p nor m was able to fold. We found that SecB bound with 100-fold higher affinity to p (Kd 0.8 nM) than it bound to m (Kd 80 nM). As the presence of the signal sequence in p is the only feature that distinguished p from m, these data strongly suggest that the high-affinity binding of SecB is to the signal sequence region and not the mature region of p. Consistent with this conclusion, we found that a wild-type signal peptide, but not an export-incompetent mutant signal peptide of another exported protein (LamB), competed for binding to p. Moreover, the high-affinity binding of SecB to p was resistant to 1 M salt, whereas the low-affinity binding of SecB to m was not. These qualitative differences suggested that SecB binding to m was primarily by electrostatic interactions, whereas SecB binding to p was primarily via hydrophobic interactions, presumably with the hydrophobic core of the signal sequence. Taken together our data strongly support the notion that SecB is primarily a specific signal-recognition factor.

Studies on the role of the hydrophobic domain of Ost4p in interactions with other subunits of yeast oligosaccharyl transferase

In the yeast, Saccharomyces cerevisiae, oligosaccharyl transferase (OT), which catalyzes the transfer of dolichol-linked oligosaccharide chains to nascent polypeptides in the endoplasmic reticulum, consists of nine nonidentical membrane protein subunits. Genetic and biochemical evidence indicated these nine proteins exist in three subcomplexes. Three of the OT subunits (Ost4p, Ost3p, and Stt3p) have been proposed to exist in one subcomplex. To investigate the interaction of these three membrane proteins, initially we carried out a mutational analysis of Ost4p, which is an extraordinarily small membrane protein containing only 36 amino acid residues. This analysis indicated that when single amino acid residues in a region close to the luminal face of the putative transmembrane domain of Ost4p were changed into an ionizable amino acid such as Lys or Asp, growth at 37°C and OT activity measured in vitro were impaired. In addition, using immunoprecipitation techniques and Western blot analysis, we found that with these mutations the interaction between Ost4p, Ost3p, and Stt3p was disrupted. Introduction of Lys or Asp residues at other positions in the putative transmembrane domain or at the N or C terminus of Ost4p had no effect on disrupting subunit interactions or impairing the activity of OT. These findings suggest that a localized region of the putative transmembrane domain of Ost4p mediates in stabilization of the interaction with the two other OT subunits (Ost3p and Stt3p) in a subcomplex in the endoplasmic reticulum membrane.