Curvature-mediated interactions between membrane proteins

Membrane proteins can deform the lipid bilayer in which they are embedded. If the bilayer is treated as an elastic medium, then these deformations will generate elastic interactions between the proteins. The interaction between a single pair is repulsive. However, for three or more proteins, we show that there are nonpairwise forces whose magnitude is similar to the pairwise forces. When there are five or more proteins, we show that the nonpairwise forces permit the existence of stable protein aggregates, despite their pairwise repulsions.     

Formation of electrostatic interactions on the protein-folding pathway

We describe a novel method of obtaining information about the structures of transient conformations on the folding pathway from their ionization equilibria: the H+ -titration behavior of a protein residue is determined in detail by its environment. We follow the consolidation of electrostatic interactions in the folding process by comparing the acid-titration behavior of four conformations on the folding pathway of barnase: the denatured state (D); the folding intermediate (I); the major transition state(+); and the native state (N) in the scheme D <==>I<==>(+)<==)N. The results show that strong electrostatic interactions are present in the major transition state: some of its carboxylate groups display the highly anomalous pKA values of <2 that are found in N. However, the network of ionic surface interactions is not formed in (+), and the overall protection of titrating residues is weakened. The results are consistent with the transition state being an expanded form of the native state, with a weakened but poorly hydrated core and a loosened periphery. The surface residues in such an expanded conformation are, on average, farther apart than are those in the center of the molecule. The results concerning the folding intermediate are less clear cut. We show that the interpretation of kinetic data relating to folding intermediates depends critically on assumptions about their equilibrium with other denatured states. We have, however, characterized the pH and ionic strength dependence of an apparent stability of I, using the deviation from two-state folding behavior, which can be used to investigate electrostatic properties of folding intermediates from a variety of mechanisms. In general, the data imply that I is somewhat similar to (+). Apparently odd titration properties of I are investigated further in the accompanying paper [Oliveberg, M., & Fersht, A. (1996) Biochemistry 35, 2738-2749]. The approach in this study may be of particular use in testing theoretical results since the relationship between H+ -titration properties and protein structure can be treated by classical electrostatics.     

Intramembrane molecular dipoles affect the membrane insertion and folding of a model amphiphilic peptide

The relationship between the dipole potential and the interaction of the mitochondrial amphipathic signal sequence known as p25 with model membranes has been studied using 1-(3-sulfonatopropyl)-4-[beta[2-(di-n-octyl-amino)-6-naphthyl]viny l] pyridinium betaine (di-8-ANEPPS) as a fluorescent probe. The dipole potential of phosphatidylcholine membranes was modified by incorporating into the bilayer the sterols phloretin and 6-ketocholestanol (KC), which decrease and increase the dipole potential, respectively. The results derived from the application of a dual-wavelength ratiometric fluorescence method for following the variation of the membrane dipole potential have shown that when p25 inserts into the lipidic bilayer, a decrease in the dipole potential takes place. The magnitude of this decrease depends on the initial value of the dipole potential, i.e., before interaction with the peptide. Thus, when KC was incorporated into the bilayer, the decrease caused by the membrane insertion of p25 was larger than that caused in PC membranes. Alternatively, in the presence of phloretin, the decrease in the potential caused by the peptide insertion was smaller. Complementary studies involving attenuated total reflectance-Fourier transform infrared spectroscopy of the peptide membrane interactions have shown that modification of the dipole potential affects the conformation of the peptide during the course of its interaction with the membrane. The presence of KC induces a higher amount of helicoidal structure. The presence of phloretin, however, does not appear to affect the secondary structure of the peptide. The differences observed in the dipole potential decreases caused by the presence of the peptide with the PC membranes and phloretin-PC membranes, therefore, must involve differences in the tertiary and, perhaps, quaternary conformations of p25.     

Kinetic role of electrostatic interactions in the unfolding of hyperthermophilic and mesophilic rubredoxins

The temperature dependence of the unfolding kinetics of rubredoxins from the hyperthermophile Pyrococcus furiosus (RdPf) and the mesophile Clostridium pasteurianum (RdCp) has been studied. Results show that RdPf unfolds much more slowly, under all experimentally accessible temperature regimes, than RdCp and other typical mesophilic proteins. Rates of RdCp and RdPf unfolding decrease upon increasing the pH above 2 and diverge dramatically at pH 7. As shown by detailed electrostatic energy calculations, this is the result of a differential degree of protonation of the negatively charged amino acids, which causes distinct electrostatic configurations as a function of pH. We propose that ion pairs, particularly those that are placed in key surface positions, may play a kinetic role by mildly clamping the protein and thereby influencing the nature and the number of the vibrational normal modes that are thermally accessible upon unfolding. More generally, these modes are also likely to be affected by the favorable electrostatic configurations, which we have shown to be directly linked to the extremely slow unfolding rates of RdPf at neutral pH. Even at pH 2, in the absence of any salt bridges, the unfolding rates of RdPf are much smaller than those of RdCp. This is ascribed to presently unidentified structural elements of nonelectrostatic nature. Since electrostatic effects influence the unfolding kinetics of both mesophilic and thermophilic rubredoxins, these findings may be of general significance for proteins.     

Characterization of interactions between the neurofilament triplet proteins by the yeast two-hybrid system

In the adult axon, the neurofilaments (NFs) are heteropolymers formed from the low (NFL), middle (NFM), and high (NFH) molecular weight neurofilament triplet proteins (NFTPs). All three proteins have the basic intermediate filament protein tripartite structure, which consists of a short amino-terminal head region, an alpha-helical rod region of approximately310 amino acids, and a carboxyl-terminal tail region of variable length. In vitro polymerization studies have shown that only NFL can assemble into homopolymeric 10-nm filaments. The assembly of intermediate filaments, including the NFs, begins with the formation of a coiled-coil dimer involving the alpha-helical rod domains of two molecules. In order to determine whether homodimers or heterodimers of NFTPs are the preferred intermediates in the assembly of NFs, we have used the yeast two-hybrid system to study the interactions between the different NFTPs. By monitoring the activity of the lacZ reporter gene product, we are able to show that the interactions of NFL with NFL, NFM, or NFH are stronger than the interactions of NFM with NFM or NFH and the interaction of NFH with NFH. These results imply that NFM and NFH are more likely to form heterodimers with NFL than homodimers and are consistent with the inability of NFM and NFH to self-polymerize in vitro and in vivo.     

Coordination of an array of signaling proteins through homo- and heteromeric interactions between PDZ domains and target proteins

The rapid activation and feedback regulation of many G protein signaling cascades raises the possibility that the critical signaling proteins may be tightly coupled. Previous studies show that the PDZ domain containing protein INAD, which functions in Drosophila vision, coordinates a signaling complex by binding directly to the light-sensitive ion channel, TRP, and to phospholipase C (PLC). The INAD signaling complex also includes rhodopsin, protein kinase C (PKC), and calmodulin, though it is not known whether these proteins bind to INAD. In the current work, we show that rhodopsin, calmodulin, and PKC associate with the signaling complex by direct binding to INAD. We also found that a second ion channel, TRPL, bound to INAD. Thus, most of the proteins involved directly in phototransduction appear to bind to INAD. Furthermore, we found that INAD formed homopolymers and the homomultimerization occurred through two PDZ domains. Thus, we propose that the INAD supramolecular complex is a higher order signaling web consisting of an extended network of INAD molecules through which a G protein-coupled cascade is tethered.     

Yeast Snc proteins complex with Sec9. Functional interactions between putative SNARE proteins

Yeast possess two homologs of the synaptobrevin family of vesicle-associated proteins that are proposed to be involved in membrane recognition and to act as receptors for components of the fusion machinery in neurons. We have previously described the yeast homologs, Snc1 and Snc2, and demonstrated that they localize to secretory vesicles and are required for normal secretion. Yeast lacking Snc protein expression accumulate post-Golgi transport vesicles that contain secretory proteins. Therefore, Snc proteins are essential for the fusion of carrier vesicles with the plasma membrane, and this property appears to have been conserved in evolution. We have now examined whether Snc proteins interact with other components of the late secretory pathway in yeast. Here we show that Snc proteins form a tight genetic and physical interaction with a plasma membrane protein, Sec9. Sec9 is the yeast equivalent of SNAP-25, a second receptor protein from neurons that has been shown to interact with synaptobrevin. We suggest, then, that recognition of the plasma membrane by secretory vesicles may involve the formation of a Snc-Sec9 complex and that this interaction has evolved as a fundamental step in secretory processes.     

Role of electrostatic interactions in defining the potency of neurotoxin B-IV from Cerebratulus lacteus

Chemical modification implicates arginine residues of the Cerebratulus lacteus neurotoxin B-IV in biological activity. In the present study, we used site-directed mutagenesis to assess the functional contributions of each of these residues. Panels of mutants at each site have been constructed by polymerase chain reaction and recombinant proteins expressed and purified to homogeneity using an Escherichia coli expression system developed in this laboratory. All substitutions for Arg-17 (Gln, Ala, or Lys) yield proteins having undetectable levels of activity, while charge neutralizing replacement of Arg-25 (R25Q) causes a 400-fold reduction in specific toxicity. However, the R25K mutein is almost as active as natural toxin. Circular dichroism spectroscopy indicates that there are no major conformational changes in any of these muteins. These results therefore demonstrate the requirement for a guanidinium group at position 17, and a positive charge at position 25. NMR analyses (Hansen, P. E., Kem, W. R., Bieber, A. L., and Norton, R. S. (1992) Eur. J. Biochem. 210, 231-240) reveal neurotoxin B-IV to contain two antiparallel alpha-helices, which together include 57% of the sequence. Both Arg-17 and Arg-25 lie on the same face of the N-terminal helix (residues 13-26), as do the carboxyl groups of Glu-13 and Asp-21. However, charge neutralizing mutations of the latter two sites have no effects on biological activity. Arg-34, situated near the N terminus of helix 2 (residues 33-49) is also important for activity, as its replacement by Gln or Ala diminishes activity by 20- and 80-fold, respectively. However, unlike Arg-17 and Arg-25, thermal denaturation experiments suggest that R34Q may be structurally destabilized relative to wild-type B-IV.     

Stimulation through the T cell receptor leads to interactions between SHB and several signaling proteins

Shb is a recently described Src homology 2 (SH2) domain-containing adaptor protein. Here we show that Shb is expressed in lymphoid tissues, and is recruited into signaling complexes upon activation of Jurkat T cells. Grb2 binds proline-rich motifs in Shb via its SH3 domains. As a result, a number of proteins detected in anti-Shb and anti-Grb2 immunoprecipitates are shared, including phosphoproteins of 22, 36/38, 55/57 and 70 kDa. Shb-association with p22, which represents the T cell receptor associated zeta chain, occurs through the Shb SH2 domain. The central region of Shb binds p36/38. Since this interaction was inhibited by phosphotyrosine, this region of Shb is likely to contain a non-SH2 PTB (phosphotyrosine binding) domain. The Shb PTB domain was found to preferentially bind the sequence Asp-Asp-X-pTyr when incubated with a phosphopeptide library. A peptide corresponding to a phosphorylation site in 34 kDa Lnk inhibited association between Shb and p36/38. Overexpression of Shb in Jurkat cells led to increased basal phosphorylation of Shb-associated p36/38 and p70 proteins. Inactivation of the Shb SH2 domain by an R522K mutation resulted in a reduced stimulation of tyrosine phosphorylation of several proteins in response to CD3 crosslinking when expressed in Jurkat cells. Together, our results show three distinct domains of Shb all participate in the formulation of multimeric signaling complexes in activated T cells. These results indicate that the Shb protein functions in T cell receptor signaling.     

Functional interactions between SV40 T antigen and other replication proteins at the replication fork

The functional interaction of simian virus 40 (SV40) large tumor antigen (T antigen) with DNA polymerase alpha (pol alpha)-primase complex, human single-stranded DNA binding protein (HSSB), and DNA polymerase delta (pol delta) holoenzyme, which includes pol delta, activator I (also called replication factor C), and proliferating cell nuclear antigen, at the replication fork was examined using the purified components that support SV40 DNA replication. Dilution of reaction mixtures during RNA primer synthesis revealed that T antigen remained associated continuously with the fork, while the pol alpha-primase complex dissociated from the complex during oligoribonucleotide synthesis. T antigen unwound duplex DNA from the SV40 core origin at a rate of 200 base pairs/min. Pol alpha-primase complex inhibited the rate of the unwinding reaction, and HSSB, pol alpha, and primase were all required for this effect. These requirements are the same as those essential for DNA primase-catalyzed oligoribonucleotide synthesis (Matsumoto, T., Eki, T., and Hurwitz, J. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 9712-9716). This result suggests that the pol alpha-primase complex interacts with T antigen and HSSB during the unwinding reaction to synthesize RNA primers and that the interaction decreases the rate of T antigen movement. While pol delta holoenzyme can elongate primed DNA chains at a rate of 400-600 nucleotides/min on singly primed phi X174 DNA, the rate of the leading strand synthesis catalyzed by pol delta holoenzyme in the SV40 replication system in vitro was about 200 nucleotides/min. This rate was similar to the unwinding rate catalyzed by T antigen. Thus, the rate of leading strand synthesis catalyzed by pol delta holoenzyme in vitro appears to be limited by the unwinding reaction catalyzed by T antigen.     

Non-proline cis peptide bonds in proteins

In a non-redundant set of 571 proteins from the Brookhaven Protein Data Base, a total of 43 non-proline cis peptide bonds were identified. Average geometrical parameters of the well-defined cis peptide bonds in proteins determined at high resolution show that some parameters, most notably the bond angle at the amide bond nitrogen, deviate significantly from the corresponding one in the trans conformation. Since the same feature was observed in cis amide bonds in small molecule structures found in the Cambridge Structural Data Base, a new set of parameters for the refinement of protein structures containing non-Pro cis peptide bonds is proposed.A striking preference was observed for main-chain dihedral angles of the residues involved in cis peptide bonds. All residues N-terminal and most residues C-terminal to a non-Pro cis peptide bond (except Gly) are located in the beta-region of a phi/psi plot. Also, all of the few C-terminal residues (except Gly) located in the alpha-region of the phi/psi plot constitute the start of an alpha-helix in the respective structure. In the majority of cases, an intimate side-chain/side-chain interaction was observed between the flanking residues, often involving aromatic side-chains. Interestingly, most of the cases found occur in functionally important regions such as close to the active site of proteins. It is intriguing that many of the proteins containing non-proline cis peptide bonds are carbohydrate-binding or processing proteins. The occurrence of these unusual peptide bonds is significantly more frequent in structures determined at high resolution than in structures determined at medium and low resolution, suggesting that these bonds may be more abundant than previously thought. On the basis of our experience with the structure determination of coagulation factor XIII, we developed an algorithm for the identification of possibly overlooked cis peptide bonds that exploits the deviations of geometrical parameters from ideality. A few likely candidates based on our algorithm have been identified and are discussed.     

Characterization of pKa values and titration shifts in the cytotoxic ribonuclease alpha-sarcin by NMR. Relationship between electrostatic interactions, structure, and catalytic function

The electrostatic behavior of titrating groups in alpha-sarcin was investigated using 1H NMR spectroscopy. A total of 209 chemical shift titration curves corresponding to different protons in the molecule were determined over the pH range of 3.0-8.5. Nonlinear least-squares fits of the data to simple relationships derived from the Henderson-Hasselbalch equation led to the unambiguous determination of pKa values for all glutamic acid and histidine residues, as well as for the C-terminal carboxylate and most of the aspartic acids in the free enzyme. The ionization constants of catalytically relevant histidines, His50 and His137, and glutamic acid, Glu96, in the alpha-sarcin-2'-GMP complex were also determined. The pKa values of 15 ionizable groups (C-carboxylate, six aspartic acids, four glutamic acids, and four histidines) were found to be close to their normal values. On the other hand, a number of side chain groups, including those in the active center, showed pKa values far from their intrinsic values. Thus, the pKa values for active site residues His50, Glu96, and His137 were 7.7, 5.2, and 5.8 in the free enzyme and 7.6, approximately 4.8, and 6.8 in the alpha-sarcin-2'-GMP complex, respectively. The pKa values and the activity profile against ApA, as a function of pH, are in agreement with the proposed enzymatic mechanism (in common with RNase T1 and the family of the microbial ribonucleases), in which Glu96 and His137 act as a general base and general acid, respectively. In almost all microbial ribonucleases, a Phe-His interaction is present, which affects the pKa of one of the His residues at the active site (His137). The absence of this interaction in alpha-sarcin would explain the lower pKa value of this His residue, and provides an explanation for the decreased RNase activity of this protein as compared to those of other microbial ribonucleases.     

Molecular interactions between the HSV-1 capsid proteins as measured by the yeast two-hybrid system

HSV-1 B capsids are composed of seven major proteins, designated VP5, VP19C, 21, 22a, VP23, VP24, and VP26. VP indicates that the capsid protein is also a component of the infectious virion. Capsid proteins 21, 22a, and VP24 are specified by a single open reading frame (UL26) that encodes 635 amino acids. An objective of the work in our laboratory is to identify and map interactions among and between capsid proteins. In the present studies we employed the yeast GAL4 two-hybrid system developed by Fields and his colleagues (Nature 240, 245-246 (1989)) for this purpose. DNA corresponding to the capsid open reading frames was derived as a PCR product and fused to sequences of the GAL4 activation and DNA binding domains. Using this system each of the capsid proteins has been tested for interactions with all of the other capsid proteins. Three interactions have been identified: a relatively strong self-interaction between 22a molecules (residues 307-635 of UL26), bimolecular interactions between 22a and VP5, and another between VP19C and VP23. The interactions were detected by the expression of beta-galactosidase enzyme activity, and yielded 289, 86, and 63 units of enzyme activity, respectively. For the 22a self-interaction, elimination of residues 611-635 resulted in an approximately twofold decrease in enzyme activity. The C-terminal 25 amino acids of 22a were also essential for the bimolecular interaction between 22a and VP5.     

pi-Stacking interactions. Alive and well in proteins

A representative set of high resolution x-ray crystal structures of nonhomologous proteins have been examined to determine the preferred positions and orientations of noncovalent interactions between the aromatic side chains of the amino acids phenylalanine, tyrosine, histidine, and tryptophan. To study the primary interactions between aromatic amino acids, care has been taken to examine only isolated pairs (dimers) of amino acids because trimers and higher order clusters of aromatic amino acids behave differently than their dimer counterparts. We find that pairs (dimers) of aromatic side chain amino acids preferentially align their respective aromatic rings in an off-centered parallel orientation. Further, we find that this parallel-displaced structure is 0.5-0.75 kcal/mol more stable than a T-shaped structure for phenylalanine interactions and 1 kcal/mol more stable than a T-shaped structure for the full set of aromatic side chain amino acids. This experimentally determined structure and energy difference is consistent with ab initio and molecular mechanics calculations of benzene dimer, however, the results are not in agreement with previously published analyses of aromatic amino acids in proteins. The preferred orientation is referred to as parallel displaced pi-stacking.