Biomedical subjects

Sanguk Kim

Publications and source records attributed to Sanguk Kim.

16 recordsLinked to original sources

PubMed2006

Janus model of the Na,K-ATPase beta-subunit transmembrane domain: distinct faces mediate alpha/beta assembly and beta-beta homo-oligomerization.

Na,K-ATPase is a hetero-oligomer of alpha and beta-subunits. The Na,K-ATPase beta-subunit (Na,K-beta) is involved in both the regulation of ion transport activity, and in cell-cell adhesion. By structure prediction and evolutionary analysis, we identified two distinct faces on the Na,K-beta transmembrane domain (TMD) that could mediate protein-protein interactions: a glycine zipper motif and a conserved heptad repeat. Here, we show that the heptad repeat face is involved in the hetero-oligomeric interaction of Na,K-beta with Na,K-alpha, and the glycine zipper face is involved in the homo-oligomerization of Na,K-beta. Point mutations in the heptad repeat motif reduced Na,K-beta binding to Na,K-alpha, and Na,K-ATPase activity. Na,K-beta TMD homo-oligomerized in biological membranes, and mutation of the glycine zipper motif affected oligomerization and cell-cell adhesion. These results provide a structural basis for understanding how Na,K-beta links ion transport and cell-cell adhesion.

Amino Acid Sequence↗

PubMed2006

Engineering a de novo internal disulfide bridge to improve the thermal stability of xylanase from Bacillus stearothermophilus No. 236.

Improvement of thermal stability of the Bacillus stearthermophilus No. 236 endo-beta-1,4-xylanase (XynA) was tried by engineering a de novo designed disulfide bridge. Disulfide design was performed firstly using the disulfide bond design program (Disulfide by Designtrade mark) to identify residue pairs having the favorable geometric characteristics for disulfide formation. Subsequently, the selected 25 amino acid pairs were filtered with the evolutionarily conserved Cys residues identified by alignment of 34 family 11 mesophilic and thermophilic xylanases, and also by doing inspection of the molecular model of the xylanases. Only one pair (Ser100 and Asn150) was finally chosen, and the respective amino acids were substituted with cysteine residues. The newly designed disulfide bridge increased thermostability of the XynA about 5 degrees C. This improved thermal stability was supported by the increase in the energy barrier for inactivation. As expected, the mutant XynA SNC demonstrated its better use in the hydrolysis of xylan at substantially higher temperatures than permitted by its native counterpart. The mutation had little influence on the catalytic efficiency and other functional properties of the XynA. In conclusion, it is evident that the strategically placed disulfide bridge has made the XynA be more effective in resisting thermal inactivation.

Amino Acid Sequence↗

PubMed2006

Self-association of the transmembrane domain of an anthrax toxin receptor.

Protective antigen (PA), lethal factor (LF) and edema factor (EF) are secreted individually by Bacillus anthracis. These components of anthrax toxin must then assemble into complexes to intoxicate mammalian cells. Toxin assembly initiates when molecules of PA bind mammalian receptors ANTXR1/2 and are cleaved by surface proteases into 20 kDa and 63 kDa fragments. After PA20 dissociates, receptor-bound PA63 homo-oligomerizes into heptamers. Oligomeric PA63 binds EF and LF and these complexes are internalized into an acidic compartment where the two enzymatic components are translocated across the membrane by a channel formed by heptameric PA63. Since oligomerization of PA63 is required to bind and translocate the enzymatic components, we sought to determine whether interactions between toxin receptors could facilitate the assembly process. In the present work, we performed a co-immunoprecipitation experiment to demonstrate that ANTXR1 is oligomeric in mammalian cells. Computer modeling predicted the self-association of the ANTXR1 transmembrane domain and we detected oligomerization of ANTXR1 transmembrane domain peptides in the membrane-mimetic environment of SDS micelles using fluorescence resonance energy transfer. Furthermore, the ANTXR1 transmembrane domain mediated oligomerization of a reporter protein construct in a bacterial membrane. In both assays, mutations that disrupted the interaction were consistent with the interaction being mediated through an asymmetric binding interface. Mutations that impaired self-association of the transmembrane domain reduced the rate of PA63 heptamer formation on the mammalian cell surface. Our findings indicate that ANTXR1 transmembrane domains self-associate and that these interactions may stabilize intermediate oligomerization states of ANTXR1-PA63 complexes.

Amino Acid Sequence↗

PubMed2006

A limited universe of membrane protein families and folds.

One of the goals of structural genomics is to obtain a structural representative of almost every fold in nature. A recent estimate suggests that 70%-80% of soluble protein domains identified in the first 1000 genome sequences should be covered by about 25,000 structures-a reasonably achievable goal. As no current estimates exist for the number of membrane protein families, however, it is not possible to know whether family coverage is a realistic goal for membrane proteins. Here we find that virtually all polytopic helical membrane protein families are present in the already known sequences so we can make an estimate of the total number of families. We find that only approximately 700 polytopic membrane protein families account for 80% of structured residues and approximately 1700 cover 90% of structured residues. While apparently a finite and reachable goal, we estimate that it will likely take more than three decades to obtain the structures needed for 90% residue coverage, if current trends continue.

Algorithms↗

PubMed2005

Functional characterization of sequence motifs in the transit peptide of Arabidopsis small subunit of rubisco.

The transit peptides of nuclear-encoded chloroplast proteins are necessary and sufficient for targeting and import of proteins into chloroplasts. However, the sequence information encoded by transit peptides is not fully understood. In this study, we investigated sequence motifs in the transit peptide of the small subunit of the Rubisco complex by examining the ability of various mutant transit peptides to target green fluorescent protein reporter proteins to chloroplasts in Arabidopsis (Arabidopsis thaliana) leaf protoplasts. We divided the transit peptide into eight blocks (T1 through T8), each consisting of eight or 10 amino acids, and generated mutants that had alanine (Ala) substitutions or deletions, of one or two T blocks in the transit peptide. In addition, we generated mutants that had the original sequence partially restored in single- or double-T-block Ala (A) substitution mutants. Analysis of chloroplast import of these mutants revealed several interesting observations. Single-T-block mutations did not noticeably affect targeting efficiency, except in T1 and T4 mutations. However, double-T mutants, T2A/T4A, T3A/T6A, T3A/T7A, T4A/T6A, and T4A/T7A, caused a 50% to 100% loss in targeting ability. T3A/T6A and T4A/T6A mutants produced only precursor proteins, whereas T2A/T4A and T4A/T7A mutants produced only a 37-kD protein. Detailed analyses revealed that sequence motifs ML in T1, LKSSA in T3, FP and RK in T4, CMQVW in T6, and KKFET in T7 play important roles in chloroplast targeting. In T1, the hydrophobicity of ML is important for targeting. LKSSA in T3 is functionally equivalent to CMQVW in T6 and KKFET in T7. Furthermore, subcellular fractionation revealed that Ala substitution in T1, T3, and T6 produced soluble precursors, whereas Ala substitution in T4 and T7 produced intermediates that were tightly associated with membranes. These results demonstrate that the transit peptide contains multiple motifs and that some of them act in concert or synergistically.

Alanine↗

PubMed2005

Transmembrane glycine zippers: physiological and pathological roles in membrane proteins.

We have observed a common sequence motif in membrane proteins, which we call a glycine zipper. Glycine zipper motifs are strongly overrepresented and conserved in membrane protein sequences, and mutations in glycine zipper motifs are deleterious to function in many cases. The glycine zipper has a significant structural impact, engendering a strong driving force for right-handed packing against a neighboring helix. Thus, the presence of a glycine zipper motif leads directly to testable structural hypotheses, particularly for a subclass of glycine zipper proteins that form channels. For example, we suggest that the membrane pores formed by the amyloid-beta peptide in vitro are constructed by glycine zipper packing and find that mutations in the glycine zipper motif block channel formation. Our findings highlight an important structural motif in a wide variety of normal and pathological processes.

Amino Acid Motifs↗

PubMed2005

Pivotal role of the glycine-rich TM3 helix in gating the MscS mechanosensitive channel.

The crystal structure of an open form of the Escherichia coli MscS mechanosensitive channel was recently solved. However, the conformation of the closed state and the gating transition remain uncharacterized. The pore-lining transmembrane helix contains a conserved glycine- and alanine-rich motif that forms a helix-helix interface. We show that introducing 'knobs' on the smooth glycine face by replacing glycine with alanine, and substituting conserved alanines with larger residues, increases the pressure required for gating. Creation of a glycine-glycine interface lowers activation pressure. The importance of residues Gly104, Ala106 and Gly108, which flank the hydrophobic seal, is demonstrated. A new structural model is proposed for the closed-to-open transition that involves rotation and tilt of the pore-lining helices. Introduction of glycine at Ala106 validated this model by acting as a powerful suppressor of defects seen with mutations at Gly104 and Gly108.

Alanine↗

PubMed2004

Transmembrane domain helix packing stabilizes integrin alphaIIbbeta3 in the low affinity state.

Regulated changes in the affinity of integrin adhesion receptors ("activation") play an important role in numerous biological functions including hemostasis, the immune response, and cell migration. Physiological integrin activation is the result of conformational changes in the extracellular domain initiated by the binding of cytoplasmic proteins to integrin cytoplasmic domains. The conformational changes in the extracellular domain are likely caused by disruption of intersubunit interactions between the alpha and beta transmembrane (TM) and cytoplasmic domains. Here, we reasoned that mutation of residues contributing to alpha/beta interactions that stabilize the low affinity state should lead to integrin activation. Thus, we subjected the entire intracellular domain of the beta3 integrin subunit to unbiased random mutagenesis and selected it for activated mutants. 25 unique activating mutations were identified in the TM and membrane-proximal cytoplasmic domain. In contrast, no activating mutations were identified in the more distal cytoplasmic tail, suggesting that this region is dispensable for the maintenance of the inactive state. Among the 13 novel TM domain mutations that lead to integrin activation were several informative point mutations that, in combination with computational modeling, suggested the existence of a specific TM helix-helix packing interface that maintains the low affinity state. The interactions predicted by the model were used to identify additional activating mutations in both the alpha and beta TM domains. Therefore, we propose that helical packing of the alpha and beta TM domains forms a clasp that regulates integrin activation.

Amino Acid Sequence↗

PubMed2004

Snorkeling preferences foster an amino acid composition bias in transmembrane helices.

By analyzing transmembrane (TM) helices in known structures, we find that some polar amino acids are more frequent at the N terminus than at the C terminus. We propose the asymmetry occurs because most polar amino acids are better able to snorkel their polar atoms away from the membrane core at the N terminus than at the C terminus. Two findings lead us to this proposition: (1) side-chain conformations are influenced strongly by the N or C-terminal position of the amino acid in the bilayer, and (2) the favored snorkeling direction of an amino acid correlates well with its N to C-terminal composition bias. Our results suggest that TM helix predictions should incorporate an N to C-terminal composition bias, that rotamer preferences of TM side-chains are position-dependent, and that the ability to snorkel influences the evolutionary selection of amino acids for the helix N and C termini.

Amino Acid Sequence↗

PubMed2004

Membrane channel structure of Helicobacter pylori vacuolating toxin: role of multiple GXXXG motifs in cylindrical channels.

Helicobacter pylori is a human pathogen responsible for severe gastric diseases such as peptic ulcers, gastric adenocarcinoma, and gastric lymphoma. Vacuolating toxin (VacA) is crucial in facilitating the colonization of the gastric lining by inducing cell apoptosis and immune suppression. VacA inserts into membranes and forms a hexameric, anion-selective pore. Here we present a structural model of the VacA pore that strongly resembles the structure of an unrelated anion-selective channel, MscS. In our model, Gly residues in GXXXG motifs pack against small Ala or Val side chains to generate the pore. Our model suggests that the same design of two anion-selective channels was achieved by two different evolutionary paths and provides insight into the mechanism of VacA function.

Amino Acid Motifs↗

PubMed2004

The affinity of GXXXG motifs in transmembrane helix-helix interactions is modulated by long-range communication.

Sequence motifs are responsible for ensuring the proper assembly of transmembrane (TM) helices in the lipid bilayer. To understand the mechanism by which the affinity of a common TM-TM interactive motif is controlled at the sequence level, we compared two well characterized GXXXG motif-containing homodimers, those formed by human erythrocyte protein glycophorin A (GpA, high-affinity dimer) and those formed by bacteriophage M13 major coat protein (MCP, low affinity dimer). In both constructs, the GXXXG motif is necessary for TM-TM association. Although the remaining interfacial residues (underlined) in GpA (LIXXGVXXGVXXT) differ from those in MCP (VVXXGAXXGIXXF), molecular modeling performed here indicated that GpA and MCP dimers possess the same overall fold. Thus, we could introduce GpA interfacial residues, alone and in combination, into the MCP sequence to help decrypt the determinants of dimer affinity. Using both in vivo TOXCAT assays and SDS-PAGE gel migration rates of synthetic peptides derived from TM regions of the proteins, we found that the most distal interfacial sites, 12 residues apart (and approximately 18 A in structural space), work in concert to control TM-TM affinity synergistically.

Amino Acid Motifs↗

PubMed2004

2D solid state NMR spectral simulation of 3(10), alpha, and pi-helices.

Transmembrane helices are more uniform in structure than similar helices in water soluble proteins. Solid state NMR of aligned bilayer samples is being increasingly used to characterize helical membrane protein structures. Traditional spectroscopic methods have difficulty distinguishing between helices with i to i + 3 (3(10)), i to i + 4 (alpha), and i to i + 5 (pi) hydrogen bonding topology. Here, we show that resonance patterns in PISEMA spectra simulated for these different helices show unique and striking features. The size and shape of these Polar Index Slant Angle (PISA) wheels, as well as the resonances per turn and clockwise versus counter-clockwise sequential connectivity of the resonances demonstrate how these different helical structures, if present as a uniform structure, will be readily distinguished, and characterized.

Computer Simulation↗

PubMed2004

A model of the closed form of the nicotinic acetylcholine receptor m2 channel pore.

The nicotinic acetylcholine receptor is a neurotransmitter-gated ion channel in the postsynaptic membrane. It is composed of five homologous subunits, each of which contributes one transmembrane helix--the M2 helix--to create the channel pore. The M2 helix from the delta subunit is capable of forming a channel by itself. Although a model of the receptor was recently proposed based on a low-resolution, cryo-electron microscopy density map, we found that the model does not explain much of the other available experimental data. Here we propose a new model of the M2 channel derived solely from helix packing and symmetry constraints. This model agrees well with experimental results from solid-state NMR, chemical reactivity, and mutagenesis experiments. The model depicts the channel pore, the channel gate, and the residues responsible for cation specificity.

Cell Membrane↗

PubMed2003

A simple method for modeling transmembrane helix oligomers.

We describe an effective procedure for modeling the structures of simple transmembrane helix homo-oligomers. The method differs from many previous approaches in that the only structural constraint we use to help select the correct model is the oligomerization state of the protein. The method involves the following steps: (1) perform 100-250 independent Monte Carlo energy minimizations of helix pairs to produce a large collection of well-packed structures; (2) filter the minimized structures to find those that are consistent with the expected symmetry of the oligomer; (3) cluster the structures that pass the symmetry filter; and (4) select a representative of the most populous cluster as the final prediction. We applied the method to the transmembrane helices of five proteins and compare our results to the available experimental data. Our predictions of glycophorin A, neu, the M2 channel and phospholamban resulted in a single model for each protein that agreed with the experimental results. In the case of erbB-2, however, we obtained three structurally distinct clusters of approximately equal sizes, so it was not possible to identify a clearly favored structure. This may reflect a real heterogeneity of packing modes for erbB-2, which is known to interact with different receptor subunits. Our method should be useful for obtaining structural models of transmembrane domains, improving our understanding of structure/function relationships for particular membrane proteins.

Calcium-Binding Proteins↗

PubMed2002

The closed state of a H+ channel helical bundle combining precise orientational and distance restraints from solid state NMR.

An interhelical distance has been precisely measured by REDOR solid-state NMR spectroscopy in the transmembrane tetrameric bundle of M2-TMP, from the M2 proton channel of the influenza A viral coat. The high-resolution structure of the helical backbone has been determined using orientational restraints from uniformly aligned peptide preparations in hydrated dimyristoylphosphatidylcholine bilayers. Here, the distance between (15)N(pi) labeled His37 and (13)C(gamma) labeled Trp41 is determined to be less than 3.9 A. Such a short distance, in combination with the known tilt and rotational orientation of the individual helices, permits not only a determination of which specific side chain pairings give rise to the interaction, but also the side chain torsion angles and restraints for the tetrameric bundle can also be characterized. The resulting proton channel structure is validated in a variety of ways. Both histidine and tryptophan side chains are oriented in toward the pore where they can play a significant functional role. The channel appears to be closed by the proximity of the four indoles consistent with electrophysiology and mutagenesis studies of the intact protein at pH 7.0 and above. The pore maintains its integrity to the N terminal side of the membrane, and at the same time, a cavity is generated that appears adequate for binding amantadine. Finally, the observation of a 2 kHz coupling in the PISEMA spectrum of (15)N(pi)His37 validates the orientation of the His37 side chain based on the observed REDOR distance.

Amino Acid Sequence↗

PubMed2002

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.

Bacteriorhodopsins↗