Structural Influences: Cholesterol,Drug, and Proton Binding to Full-Length Influenza A M2 Protein

The structure and functions of the M2 protein from Influenza A are sensitive to pH, cholesterol, and the antiinfluenza drug Amantadine. This is a tetrameric membrane protein of 97 amino-acid residues that has multiple functions, among them as a proton-selective channel and facilitator of viral budding, replacing the need for the ESCRT proteins that other viruses utilize. Here, various amino-acid-specific-labeled samples of the full-length protein were prepared and mixed, so that only interresidue ¹³C-¹³C cross peaks between two differently labeled proteins representing interhelical interactions are observed. This channel is activated at slightly acidic pH values in the endosome when the His³⁷ residues in the middle of the transmembrane domain take on a +2 or +3 charged state. Changes observed here in interhelical distances in the N-terminus can be accounted for by modest structural changes, and no significant changes in structure were detected in the C-terminal portion of the channel upon activation of the channel. Amantadine, which blocks proton conductance by binding in the aqueous pore near the N-terminus, however, significantly modifies the tetrameric structure on the opposite side of the membrane. The interactions between the juxtamembrane amphipathic helix of one monomer and its neighboring monomer observed in the absence of drug are disrupted in its presence. However, the addition of cholesterol prevents this structural disruption. In fact, strong interactions are observed between cholesterol and residues in the amphipathic helix, accounting for cholesterol binding adjacent to a native palmitoylation site and near to an interhelix crevice that is typical of cholesterol binding sites. The resultant stabilization of the amphipathic helix deep in the bilayer interface facilitates the bilayer curvature that is essential for viral budding.     

Uniformity,ideality, and hydrogen bonds in transmembrane alpha-helices

Protein environments substantially influence the balance of molecular interactions that generate structural stability. Transmembrane helices exist in the relatively uniform low dielectric interstices of the lipid bilayer, largely devoid of water and with a very hydrophobic distribution of amino acid residues. Here, through an analysis of bacteriorhodopsin crystal structures and the transmembrane helix structure from M2 protein of influenza A, some helices are shown to be exceptionally uniform in hydrogen bond geometry, peptide plane tilt angle, and backbone torsion angles. Evidence from both the x-ray crystal structures and solid-state NMR structure suggests that the intramolecular backbone hydrogen bonds are shorter than their counterparts in water-soluble proteins. Moreover, the geometry is consistent with a dominance of electrostatic versus covalent contributions to these bonds. A comparison of structure as a function of resolution shows that as the structures become better characterized the helices become much more uniform, suggesting that there is a possibility that many more uniform helices will be observed, even among the moderate resolution membrane protein structures that are currently in the Protein Data Bank that do not show such features.     

Characterization and modification of a monomeric mutant of the lactose repressor protein

A monomeric mutant lactose repressor protein (T-41), containing serine at position 282 in place of tyrosine [Schmitz, A., Schmeissner, U., Miller, J. H., & Lu, P. (1976) J. Biol. Chem. 251, 3359-3366], has been purified by a series of chromatographic and precipitation methods. The molecular weight of the mutant as determined by gel filtration was approximately 40,000. The inducer equilibrium binding constant for the mutant was comparable to that of the tetrameric wild-type repressor at pH 7.5, whereas operator DNA binding was not detectable. In contrast to wild-type repressor, equilibrium and kinetic rate constants for inducer binding to the monomer were largely independent of pH; thus, the quaternary structure of the wild-type repressor is required for the pH-associated effects on inducer binding. Although ultraviolet absorbance difference spectra indicated that inducer binding to T-41 protein elicited different changes in the environment of aromatic residues from those generated in wild-type repressor, the shift in the fluorescence emission maximum in response to inducer binding was similar for T-41 and wild-type repressors. Similarity in 1-anilinonaphthalene-8-sulfonic acid binding to monomer and tetramer suggests that this fluorophore does not bind at subunit interfaces. Modification of Cys-281 with methyl methanethiosulfonate was observed at low molar ratios of reagent per T-41 monomer (4-fold). This result is in contrast to data observed for tetrameric wild-type repressor which requires high molar ratios for this cysteine to react. We conclude that Cys-281, adjacent to the site of the T-41 mutation, is located on the surface of the monomer in this region crucial for subunit interaction.     

Crystallisation and preliminary analysis of glucose isomerase from Streptomyces albus

The glucose isomerase of Streptomyces albus has been crystallised from a dilute solution of magnesium chloride buffered at a pH of 6.8-7.0. The crystals are in the space group I222 with cell dimensions a = 93.9 A, b = 99.5 A and c = 102.9 A. There is one monomer of the tetrameric molecule per asymmetric unit of the crystal and the packing density is 2.93 A3.Da-1. The tetramer sits on the 222 symmetry point of the crystal. Native data have been recorded to a resolution of 1.9 A and the crystals diffract to about 1.5 A. The alpha-carbon coordinates of the Arthrobacter glucose isomerase and the backbone coordinates of the S. olivochromogenes enzyme determined by other groups have been oriented in the present cell. The structure is currently being refined. The binding of several metal ions to the two metal sites has been analysed.     

Structural Basis of pH Dependence of Neoculin,a Sweet Taste-Modifying Protein

Among proteins utilized as sweeteners, neoculin and miraculin are taste-modifying proteins that exhibit pH-dependent sweetness. Several experiments on neoculin have shown that His11 of neoculin is responsible for pH dependence. We investigated the molecular mechanism of the pH dependence of neoculin by molecular dynamics (MD) calculations. The MD calculations for the dimeric structures of neoculin and His11 mutants showed no significant structural changes for each monomer at neutral and acidic pH levels. The dimeric structure of neoculin dissociated to form isolated monomers under acidic conditions but was maintained at neutral pH. The dimeric structure of the His11Ala mutant, which is sweet at both neutral and acidic pH, showed dissociation at both pH 3 and 7. The His11 residue is located at the interface of the dimer in close proximity to the Asp91 residue of the other monomer. The MD calculations for His11Phe and His11Tyr mutants demonstrated the stability of the dimeric structures at neutral pH and the dissociation of the dimers to isolated monomers. The dissociation of the dimer caused a flexible backbone at the surface that was different from the dimeric interface at the point where the other monomer interacts to form an oligomeric structure. Further MD calculations on the tetrameric structure of neoculin suggested that the flexible backbone contributed to further dissociation of other monomers under acidic conditions. These results suggest that His11 plays a role in the formation of oligomeric structures at pH 7 and that the isolated monomer of neoculin at acidic pH is responsible for sweetness.     

Comparison of human and shirbot (Cyprinidae: Barbus grypus) hemoglobin: a structure-function prospective

Hemoglobin is a tetrameric protein with two alpha and two beta subunits binds oxygen in a cooperative manner. In dominant tetrameric form of fish hemoglobin carry more than 90 percent of oxygen from gill to tissues at 20° C. The tetrameric form of fish hemoglobin is changed to monomeric form at low oxygen pressure in order to increase its oxygen affinity. This is one of adaptive mechanisms used by different kinds of fish. The major aim of this paper is to study the molecular basis of shirbot hemoglobin adaptation mechanism to various environmental conditions. Using different methods such as ion exchange chromatography, UV-Vis, fluorescence and circular dichroism spectroscopy, we extracted the main tetrameric fraction of shirbot hemoglobin and studied the structural characteristics of shirbot and human hemoglobins in a comparative way. Our results showed that tetrameric form of shirbot hemoglobin has less stable and loosely folded structure in contrast to human hemoglobin. Our data also indicate, in case of exposure to life-threatening environmental factors such as low oxygen level, acidic pH, oxidizing chemicals and other water pollutants especially detergents (surfactants) triggering tetramer to monomer dissociation in shirbot hemoglobin is more prominently than in human hemoglobin. The resulting monomer of hemoglobin has more oxygen affinity and could take up oxygen more strongly even at low pressure. We hypothesize that this mechanism helps shirbot to adapt and to survive at such harsh environment. The mechanism that is may be adapted by other fish species.     

Molecular heterogeneity of the concanavalin A tetramer: effects on binding to human red blood cells

We have found that the distribution of the three main monomer species found in tetrameric concanavalin A was approximately 73% type A monomer (27,000 MW); 4% type B monomer (14,000 MW); and 23% type C monomer (12,000 MW). When this tetrameric concanavalin A was bound to human erythrocytes and the monomer distribution of the bound concanavalin A was examined, we found that it resembled that of the concanavalin A used in the binding reaction. However, when competing sugars were used, either to inhibit the binding of concanavalin A or to remove previously-bound lectin, examination of cell-bound monomer distribution revealed that there was a significant increase in type C monomers and a simultaneous decrease in type A monomers. The shifts in monomer distribution varied depending on experimental conditions and the particular competing inhibitor employed. These findings were taken to indicate that not all concanavalin A cell surface interactions are identical and that quantitative methods are available for studying this phenomenon.     

Engineered streptavidin monomer and dimer with improved stability and function

Although streptavidin's high affinity for biotin has made it a widely used and studied binding protein and labeling tool, its tetrameric structure may interfere with some assays. A streptavidin mutant with a simpler quaternary structure would demonstrate a molecular-level understanding of its structural organization and lead to the development of a novel molecular reagent. However, modulating the tetrameric structure without disrupting biotin binding has been extremely difficult. In this study, we describe the design of a stable monomer that binds biotin both in vitro and in vivo. To this end, we constructed and characterized monomers containing rationally designed mutations. The mutations improved the stability of the monomer (increase in T(m) from 31 to 47 °C) as well as its affinity (increase in K(d) from 123 to 38 nM). We also used the stability-improved monomer to construct a dimer consisting of two streptavidin subunits that interact across the dimer-dimer interface, which we call the A/D dimer. The biotin binding pocket is conserved between the tetramer and the A/D dimer, and therefore, the dimer is expected to have a significantly higher affinity than the monomer. The affinity of the dimer (K(d) = 17 nM) is higher than that of the monomer but is still many orders of magnitude lower than that of the wild-type tetramer, which suggests there are other factors important for high-affinity biotin binding. We show that the engineered streptavidin monomer and dimer can selectively bind biotinylated targets in vivo by labeling the cells displaying biotinylated receptors. Therefore, the designed mutants may be useful in novel applications as well as in future studies in elucidating the role of oligomerization in streptavidin function.     

Solid-state 13C NMR spectroscopy of a 13C carbonyl-labeled polypeptide

High resolution structural elucidation of macromolecular structure by solid-state nuclear magnetic resonance requires the preparation of uniformly aligned samples that are isotopically labeled. In addition, to use the chemical shift interaction as a high resolution constraint requires an in situ tensor characterization for each site of interest. For ¹³C in the peptide backbone, this characterization is complicated by the presence of dipolar coupled ¹⁴N from the peptide bond. Here the ¹³C₁-Gly₂ site in gramicidin A is studied both as a dry powder and in a fully hydrated lipid bilayer environment. Linewidths reported for the oriented samples are a factor of five narrower than those reported elsewhere, and previous misinterpretations of the linewidths are corrected. The observed frequency from oriented samples is shown to be consistent with the recently determined structure for this site in the gramicidin backbone. It is also shown that, whereas a dipolar coupling between ¹³C and ¹⁴N is apparent in dry preparations of the polypeptide, in a hydrated bilayer the dipolar coupling is absent, presumably due to a `self-decoupling' mechanism.     

Macromolecular structural elucidation with solid-state NMR-derived orientational constraints

Summary The complete structure determination of a polypeptide in a lipid bilayer environment is demonstrated built solely upon orientational constraints derived from solid-state NMR observations. Such constraints are obtained from isotopically labeled samples uniformly aligned with respect to the B₀ field. Each observation constrains the molecular frame with respect to B₀ and the bilayer normal, which are arranged to be parallel. These constraints are not only very precise (a few tenths of a degree), but also very accurate. This is clearly demonstrated as the back bone structure is assembled sequentially and the i to i+6 hydrogen bonds in this structure of the gramicidin channel are shown on average to be within 0.5 Å of ideal geometry. Similarly, the side chains are assembled independently and in a radial direction from the backbone. The lack of considerable atomic overlap between side chains also demonstrates the accuracy of the constraints. Through this complete structure, solid-state NMR is demonstrated as an approach for determining three-dimensional macromolecular structure.     

Molecular Dynamics Simulations Reveal the HIV-1 Vpu Transmembrane Protein to Form Stable Pentamers

The human immunodeficiency virus type I (HIV-1) Vpu protein is 81 residues long and has two cytoplasmic and one transmembrane (TM) helical domains. The TM domain oligomerizes to form a monovalent cation selective ion channel and facilitates viral release from host cells. Exactly how many TM domains oligomerize to form the pore is still not understood, with experimental studies indicating the existence of a variety of oligomerization states. In this study, molecular dynamics (MD) simulations were performed to investigate the propensity of the Vpu TM domain to exist in tetrameric, pentameric, and hexameric forms. Starting with an idealized α-helical representation of the TM domain, a thorough search for the possible orientations of the monomer units within each oligomeric form was carried out using replica-exchange MD simulations in an implicit membrane environment. Extensive simulations in a fully hydrated lipid bilayer environment on representative structures obtained from the above approach showed the pentamer to be the most stable oligomeric state, with interhelical van der Waals interactions being critical for stability of the pentamer. Atomic details of the factors responsible for stable pentamer structures are presented. The structural features of the pentamer models are consistent with existing experimental information on the ion channel activity, existence of a kink around the Ile17, and the location of tetherin binding residues. Ser23 is proposed to play an important role in ion channel activity of Vpu and possibly in virus propagation.     

Proline Substitution of Dimer Interface β-strand Residues as a Strategy for the Design of Functional Monomeric Proteins

Proteins that exist in monomer-dimer equilibrium can be found in all organisms ranging from bacteria to humans; this facilitates fine-tuning of activities from signaling to catalysis. However, studying the structural basis of monomer function that naturally exists in monomer-dimer equilibrium is challenging, and most studies to date on designing monomers have focused on disrupting packing or electrostatic interactions that stabilize the dimer interface. In this study, we show that disrupting backbone H-bonding interactions by substituting dimer interface β-strand residues with proline (Pro) results in fully folded and functional monomers, by exploiting proline’s unique feature, the lack of a backbone amide proton. In interleukin-8, we substituted Pro for each of the three residues that form H-bonds across the dimer interface β-strands. We characterized the structures, dynamics, stability, dimerization state, and activity using NMR, molecular dynamics simulations, fluorescence, and functional assays. Our studies show that a single Pro substitution at the middle of the dimer interface β-strand is sufficient to generate a fully functional monomer. Interestingly, double Pro substitutions, compared to single Pro substitution, resulted in higher stability without compromising native monomer fold or function. We propose that Pro substitution of interface β-strand residues is a viable strategy for generating functional monomers of dimeric, and potentially tetrameric and higher-order oligomeric proteins.     

Backbone structure of the amantadine-blocked trans-membrane domain M2 proton channel from Influenza A virus

Amantadine is known to block the M2 proton channel of the Influenza A virus. Here, we present a structure of the M2 trans-membrane domain blocked with amantadine, built using orientational constraints obtained from solid-state NMR polarization-inversion-spin-exchange-at-the-magic-angle experiments. The data indicates a kink in the monomer between two helical fragments having 20 degrees and 31 degrees tilt angles with respect to the membrane normal. This monomer structure is then used to construct a plausible model of the tetrameric amantadine-blocked M2 trans-membrane channel. The influence of amantadine binding through comparative cross polarization magic-angle spinning spectra was also observed. In addition, spectra are shown of the amantadine-resistant mutant, S31N, in the presence and absence of amantadine.     

Structure and dynamics of the beta-barrel of the membrane transporter BtuB by site-directed spin labeling

Site-directed spin labeling and EPR spectroscopy were used to map two consecutive beta-strands of the putative transmembrane beta-barrel of BtuB. For these studies, a series of 29 consecutive single cysteine mutants of BtuB were produced covering residues 148-176. The proteins were then expressed, reacted with a sulfhydryl-specific spin label, purified in octyl glucoside (OG), and reconstituted into palmitoyloleoylphosphatidylcholine (POPC) bilayers. The labeled residues spanned from the extracellular region (position 148) to the small periplasmic loop (positions 160-163) and back up to the extracellular side (position 176) of BtuB. Continuous wave power saturation in the presence of oxygen or NiAA yielded an i, i + 2 periodicity for the collision frequencies at these sites and demonstrated the presence of a beta-strand structural motif. For both strands studied, the even-numbered residues were found to be exposed to the hydrophobic phase of the bilayer, whereas the odd-numbered residues pointed toward the interior of the barrel and the core of the protein. In addition, the collision parameters yielded the position of the protein within the bilayer. The phase relationship between the oxygen and metal collision frequencies along with the corresponding membrane depth parameters, Phi, indicates that segments 151-159 and 164-172 are within the bilayer. In POPC bilayers, there is a mobility gradient for spin labels along the barrel indicating enhanced backbone flexibility toward the periplasmic surface of the barrel. In POPC/OG mixed micelles, the even-numbered residues facing the hydrocarbon show an increased mobility compared with the bilayer environment whereas the inward-facing side chains show little change in motion. The data indicate that the protein core remains folded in POPC/OG mixed micelles but that this environment increases the backbone fluctuations of the strands. A model for the beta-barrel of BtuB is presented in part on the basis of these EPR data.     

Fourier transform infrared spectroscopic studies of the interaction of the antimicrobial peptide gramicidin S with lipid micelles and with lipid monolayer and bilayer membranes

We have utilized Fourier transform infrared spectroscopy to study the interaction of the antimicrobial peptide gramicidin S (GS) with lipid micelles and with lipid monolayer and bilayer membranes as a function of temperature and of the phase state of the lipid. Since the conformation of GS does not change under the experimental conditions employed in this study, we could utilize the dependence of the frequency of the amide I band of the central beta-sheet region of this peptide on the polarity and hydrogen-bonding potential of its environment to probe GS interaction with and location in these lipid model membrane systems. We find that the GS is completely or partially excluded from the gel states of all of the lipid bilayers examined in this study but strongly partitions into lipid micelles, monolayers, or bilayers in the liquid-crystalline state. Moreover, in general, the penetration of GS into zwitterionic and uncharged lipid bilayer coincides closely with the gel to liquid-crystalline phase transition of the lipid. However, GS begins to penetrate into the gel-state bilayers of anionic phospholipids prior to the actual chain-melting phase transition, while in cationic lipid bilayers, GS does not partition strongly into the liquid-crystalline bilayer until temperatures well above the chain-melting phase transition are reached. In the liquid-crystalline state, the polarity of the environment of GS indicates that this peptide is located primarily at the polar/apolar interfacial region of the bilayer near the glycerol backbone region of the lipid molecule. However, the depth of GS penetration into this interfacial region can vary somewhat depending on the structure and charge of the lipid molecule. In general, GS associates most strongly with and penetrates most deeply into more disordered bilayers with a negative surface charge, although the detailed chemical structure of the lipid molecule and physical organization of the lipid aggregate (micelle versus monolayer versus bilayer) also have minor effects on these processes.     

On the structure of the peptidoglycan of cell walls from Myxobacter AL-1 (myxobacterales).

Basically the peptidoglycan of Myxobater AL-1 consists of alternating beta-1,4-linked N-acetylglucosamic-N-acetylmuramic acid chains. After splitting the aminosugar backbone with a specific algal enzyme three subunits arise: a monomer, a dimer and a timer. Investigation of the monomer with specific enzymes and comparison of the degradation products to standards derived from other bacterial peptidoglycans suggest the following structure of the monomer peptide: L-alanyl-D-glutamic-L-meso-diaminopimelic-D-alanine. A D-alanyl-D-meso-diaminopimelic acid bond is the bridgebond between the peptides of the subunits.     

Stability of a melittin pore in a lipid bilayer: a molecular dynamics study

We have investigated the configuration and the stability of a single membrane pore bound by four melittin molecules and embedded in a fully hydrated bilayer lipid membrane. We used molecular dynamics simulations up to 5.8 ns. It is found that the initial tetrameric configuration decays with increasing time into a stable trimer and one monomer. This continuous transformation is accompanied by a lateral expansion of the aqueous pore exhibiting a final size comparable to experimental findings. The expansion-induced formation of an interface between the pore-lining acyl chains of the lipids and the pore water ("hydrophobic pore") is transformed into an energetically more favorable toroidal pore structure where some lipid heads are translocated from the rim to the central part of the interface ("hydrophilic pore"). The expansion of the pore is supported by the electrostatic repulsion among the alpha-helices. It is hypothesized that pore growth, and hence cell lysis, is induced by a melittin-mediated line tension of the pore.     

A low-resolution 3D model of the tetrameric alcohol dehydrogenase from Sulfolobus solfataricus

We describe the computation of a model of the thermophilic NAD-dependent homotetrameric alcohol dehydrogenase from the archaeon Sulfolobus solfataricus (SsADH). Modeling is based on the knowledge that each monomer contains two Zn ions with catalytic and structural function, respectively. In the database of known structures, proteins with similar functions are either dimers containing two zinc ions per monomer or tetramers with one zinc ion per monomer. In any case, the sequence identity of the target to the possible templates is low. A threading procedure is therefore developed which includes constraints taking into account residue conservation both at the zinc ion binding and at the monomer-monomer interaction sites in the tetrameric unit. The model is consistent with previously reported data. Furthermore, cross-linking experiments are described which support the computed tetrameric model.     

Outer surface modification of synthetic multifunctional pores

The characteristics of pores formed by p-octiphenyl beta-barrels with LWV triads at the outer surface are reported in comparison with the conventional rigid-rod beta-barrels with all-L outer surface. Maintained multifunctionality of tetrameric pores with external LWV triads (inversion of ion selectivity, molecular recognition and transformation) is implicative for intact barrel interior. Increased pore activity supports dominance of high bilayer affinity for W over low affinity for V. Transmembrane p-octiphenyl orientation (from fluorescence depth quenching) supports barrel-stave (rather than toroidal) pores and dominance of transmembrane preference of rigid rods over interfacial preference of W. Destabilization of beta-barrel pores in membranes (from short single-channel lifetimes) and in the media (from 4th-power dependence on monomer concentration) by LWV triads supports dominance of low beta-propensity for W over high beta-propensity for V. The relation between the stability of supramolecular (pre)pores and dependence of activity on monomer concentration is discussed in a more general context.