Difference: RecentReprintsQ107 (3 vs. 4)

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Preprints (publication date not yet finalized)

Phan AT, Kuryavyi V, Burge S, Neidle S, Patel DJ 'Structure of an Unprecedented G-Quadruplex Scaffold in the Human c-kit Promoter.' 2007 J Am Chem Soc 129; 4386-4392

The c-kit oncogene is an important target in the treatment of gastrointestinal tumors. A potential approach to inhibition of the expression of this gene involves selective stabilization of G-quadruplex structures that may be induced to form in the c-kit promoter region. Here we report on the structure of an unprecedented intramolecular G-quadruplex formed by a G-rich sequence in the c-kit promoter in K+ solution. The structure represents a new folding topology with several unique features. Most strikingly, an isolated guanine is involved in G-tetrad core formation, despite the presence of four three-guanine tracts. There are four loops: two single-residue double-chain-reversal loops, a two-residue loop, and a five-residue stem-loop, which contain base-pairing alignments. This unique structural scaffold provides a highly specific platform for the future design of ligands specifically targeted to the promoter DNA of c-kit.

Valentine ER, Ferrage F, Massi F, Cowburn D, and Palmer AG 'Joint Composite-Rotation Adiabatic-Sweep Isotope Filtration' J. Biomol. NMR 38 11-22.

Joint composite-rotation adiabatic-sweep isotope filter are derived by combining the composite rotation [A. C. Stuart, K. A. Borzilleri, J. M. Withka, and A. G. Palmer, J. Am. Chem. Soc. 121, 5346-5347 (1999)] and adiabatic-sweep [C. Zwahlen, P. Legault, S. J. F. Vincent, J. Greenblatt, R. Konrat, and L. E. Kay, J. Am. Chem. Soc. 119, 6711-6721 (1997); Ē. Kupče and R. Freeman, J. Magn. Reson. 127, 36-48 (1997)] approaches. The joint isotope filters have improved broadband filtration performance, even for extreme values of the one-bond 1H-13C scalar coupling constants in proteins and RNA molecules. An average Hamiltonian analysis is used to describe evolution of the heteronuclear scalar coupling interaction during the adiabatic sweeps within the isotope filter sequences. The new isotope filter elements permit improved selective detection of NMR resonance signals originating from 1H spins attached to an unlabeled natural abundance component of a complex in which other components are labeled with 13C and 15N isotopes.

Kempf, J. G.; Jung, J-y.; Ragain, C.; Sampson, N. S.; Loria, J. P. Dynamic Requirements for a functional protein hinge. 2007 J. Mol. Biol 368: 131-149

The enzyme triosephosphate isomerase (TIM) is a model of catalytic efficiency. The 11-residue loop 6 at the TIM active site plays a major role in this enzymatic prowess. The loop moves between open and closed states, which facilitate substrate access and catalysis, respectively. The N- and C-terminal hinges of loop 6 control this motion. Here, we detail flexibility requirements for hinges in a comparative solution NMR study of wild-type (WT) TIM and a quintuple mutant (PGG/GGG). The latter contained glycine substitutions in the N-terminal hinge at Val167 and Trp168, which follow the essential Pro166, and in the C-terminal hinge at Lys174, Thr175, and Ala176. Previous work demonstrated that PGG/GGG has a 10- fold higher Km value and 103-fold reduced kcat relative to WT with either [D]-glyceraldehyde 3-phosphate or dihyrdroxyacetone phosphate as substrate. Our NMR results explain this in terms of altered loop-6 dynamics in PGG/GGG. In the mutant, loop 6 exhibits conformational heterogeneity with corresponding motional rates < 750 s–1 that are an order of magnitude slower than the natural WT loop-6 motion. At the same time, ns-timescale motions of loop 6 are greatly enhanced in the mutant relative to WT. These differences from WT behavior occur in both apo PGG/GGG and in the form bound to the reactionintermediate analog, 2-phosphoglycolate (2-PGA). In addition, as indicated by 1H, 15N and 13CO chemicalshifts, the glycine substitutions (a) diminished the enzyme’s response to ligand, and (b) induced structural perturbations in apo and 2-PGA-bound forms of TIM that are atypical of those observed in WT. Altogether, these data show that PGG/GGG exists in multiple conformations that are not fully competent for ligand binding or catalysis. These experiments elucidate an important principle of catalytic hinge design in proteins: structural rigidity is essential for focused motional freedom of active-site loops.

Articles published in the last few months.

• Apr 2007

Martinez-Hackert, E, Hendrickson, WA. Structures of and interactions between domains of trigger factor from Thermotoga maritima. Acta Crystallogr D Biol Crystallogr 63: 536-47

• Mar 2007

Vorobiev SM, Neely H, Seetharaman J, Ma LC, Xiao R, Acton TB, Montelione GT, Tong L. Crystal structure of AGR_C_4470p from Agrobacterium tumefaciens. Protein Sci 16: 535-8

Townley, R, Shapiro, L. Crystal Structures of the Adenylate Sensor from Fission Yeast AMP-Activated Protein Kinase. Science 315: 1726-1729

The 5’-AMP (adenosine monophosphate)-activated protein kinase (AMPK) coordinates metabolic function with energy availability by responding to changes in intracellular ATP (adenosine triphosphate) and AMP levels. Here we report crystal structures at 2.6 and 2.9 Å resolution for ATP- and AMP-bound forms of a core αβγ adenylate-binding domain from the fission yeast AMPK homologue. ATP and AMP bind competitively to a single site in the γ subunit, with their respective phosphate groups positioned near function-impairing mutants. Surprisingly, ATP binds without counter ions, amplifying its electrostatic effects on a critical regulatory region where all three subunits converge.

• Feb 2007

Bai Y, Auperin TC, Tong L 'The use of in situ proteolysis in the crystallization of murine CstF -77.' Acta Crystallograph Sect F Struct Biol Cryst Commun; 63, 135-8

The cleavage-stimulation factor (CstF) is required for the cleavage of the 30-end of messenger RNA precursors in eukaryotes. During structure determination of the 77 kDa subunit of the murine CstF complex (CstF -77), it was serendipitously discovered that a solution infected by a fungus was crucial for the crystallization of this protein. CstF -77 was partially proteolyzed during crystallization; this was very likely to have been catalyzed by a protease secreted by the fungus. It was found that the fungal protease can be replaced by subtilisin and this in situ proteolysis protocol produced crystals of sufficient size for structural studies. After an extensive search, it was found that 55% glucose can be used as a cryoprotectant while maintaining the diffraction quality of the crystals; most other commonly used cryoprotectants were detrimental to the diffraction quality.

Rodriguez FA, Cai Y, Lin C, Tang Y, Kolbanovskiy A, Amin S, Patel DJ, Broyde S, and Geacintov, N.E. 'Exocyclic amino groups of flanking guanines govern sequence-dependent adduct conformations and local structural distortions for minor groove-aligned benzo[a]pyrenyl-guanine lesions in a GG mutation hotspot context.' Nucleic Acids Res 35 1555–1568

The environmental carcinogen benzo[a]pyrene (BP) is metabolized to reactive diol epoxides that bind to cellular DNA by predominantly forming N2-guanine adducts (G*). Mutation hotspots for these adducts are frequently found in 5'- ...GG... dinucleotide sequences, but their origins are poorly understood. Here we used high resolution NMR and molecular dynamics simulations to investigate differences in G* adduct conformations in 5'- ...CG*GC... and 5'-...CGG*C... sequence contexts in otherwise identical 12-mer duplexes. The BP rings are positioned 50 along the modified strand in the minor groove in both cases. However, subtle orientational differences cause strong distinctions in structural distortions of the DNA duplexes, because the exocyclic amino groups of flanking guanines on both strands compete for space with the BP rings in the minor groove, acting as guideposts for placement of the BP. In the 50- ... CGG*C... case, the 50-flanking G!C base pair is severely untwisted, concomitant with a bend deduced from electrophoretic mobility. In the 50- ...CG*GC... context, there is no untwisting, but there is significant destabilization of the 50-flanking Watson–Crick base pair. The minor groove width opens near the lesion in both cases, but more for 50- ...CGG*C.... Differential sequence-dependent removal rates of this lesion result and may contribute to the mutation hotspot phenomenon.

Goldbourt A, Gross BJ, Day LA, McDermott AE 'Filamentous Phage Studied by Magic-Angle Spinning NMR: Resonance Assignment and Secondary Structure of the Coat Protein in Pf1.' J Am Chem Soc 2007 129, 2338-2344

Assignments are presented for resonances in the magic-angle spinning solid-state NMR spectra of the major coat protein subunit of the filamentous bacteriophage Pf1. NMR spectra were collected on uniformly 13C and 15N isotopically enriched, polyethylene glycol precipitated samples of fully infectious and hydrated phage. Site-specific assignments were achieved for 231 of the 251 labeled atoms (92%) of the 46-residue-long coat protein, including 136 of the 138 backbone atoms, by means of two- and three-dimensional 15N and 13C correlation experiments. A single chemical shift was observed for the vast majority of atoms, suggesting a single conformation for the 7300 subunits in the 36 MDa virion in its hightemperature form. On the other hand, multiple chemical shifts were observed for the Cα, Cβ, and Cγ atoms of T5 in the helix terminus and the Cα and Cβ atoms of M42 in the DNA interaction domain. The chemical shifts of the backbone atoms indicate that the coat protein conformation involves a 40-residue continuous R-helix extending from residue 6 to the C-terminus.

Comment: This paper marks a major technical advance in assigning in the solid state NMR spectra of the 46-residue major coat protein of a bacteriophage, in its natural state, in whole phage. The biological co-author (Loren Day, Public Health Research Institute) deserves credit for his persistent ( > 20 y?) belief that NMR could tell him something about phase - a belief now realized. - David Cowburn - 8 Mar 2007

Poget SF, Cahill SM, Girvin ME 'Isotropic Bicelles Stabilize the Functional Form of a Small Multidrug-Resistance Pump for NMR Structural Studies.' J Am Chem Soc 129, 2432-2433

Although detergent micelles are commonly used as membrane mimetics in biophysical studies of membrane proteins, they do not ideally reproduce the native environment of the protein in the membrane and may fail to support the native fold of the protein. Not surprisingly, these nonphysiological conformations are particularly common for helical membrane proteins that undergo significant conformational changes as part of their functional cycle, such as the voltage sensor domain of potassium channels, G-proteincoupled receptors, and bacterial multidrug transporters. One dramatic example is the small multidrug-resistance pump EmrE from Escherichia coli, which was found in three significantly different conformations in two X-ray structures in detergent^1,2 and a cryo-electron microscopy 3D reconstruction map from 2D crystals.³ The functional relevance of these structures is the subject of current debate,⁴ and an alternative method of getting highresolution structural information about the functional protein is certainly needed. Bicelles, which are a mixture of short- and longchain lipids, have long been suggested as a more native-like solubilizing agent for the study of membrane proteins,^5-7 and have been used in a number of biophysical and structural studies of these proteins (for a recent review, see ref 8). Here we show that bicelles are a promising system in the study of Smr, the EmrE homologue from Staphylococcus aureus, since they both preserve the ligandbinding activity and produce NMR spectra that allowed making substantial NMR backbone assignments for this conformationally flexible protein.

Comment: Verification of how membrane proteins really fold in model systems of detergents, or after any isolation from their original membrane, is a key issue, which is still in confusion. Girvin et al. demonstrate two major points -- a solubilization medium which preserves function of the protein, and feasibility of substantial assignment for structure / function correlation by NMR in solution.- David Cowburn - 28 Feb 2007

• Jan 2007

Jingshi Shen, David C. Tareste, Fabienne Paumet, James E. Rothman and Thomas J. Melia, 'Selective Activation of Cognate SNAREpins by Sec1/Munc18 Proteins' Cell 128 183-195

Zheng Q, Deng Y, Liu J, Hoek LV, Berkhout B, Lu M 'Core Structure of S2 from the Human Coronavirus NL63 Spike Glycoprotein(,).' Biochemistry 45: 15205-15215

Human coronavirus NL63 (HCoV-NL63) has recently been identified as a causative agent of acute respiratory tract illnesses in infants and young children. The HCoV-NL63 spike (S) protein mediates virion attachment to cells and subsequent fusion of the viral and cellular membranes. This viral entry process is a primary target for vaccine and drug development. HCoV-NL63 S is expressed as a singlechain glycoprotein and consists of an N-terminal receptor-binding domain (S1) and a C-terminal transmembrane fusion domain (S2). The latter contains two highly conserved heptad-repeat (HR) sequences that are each extended by 14 amino acids relative to those of the SARS coronavirus or the prototypic murine coronavirus, mouse hepatitis virus. Limited proteolysis studies of the HCoV-NL63 S2 fusion core identify an R-helical domain composed of a trimer of the HR segments N57 and C42. The crystal structure of this complex reveals three C42 helices entwined in an oblique and antiparallel manner around a central triple-stranded coiled coil formed by three N57 helices. The overall geometry comprises distinctive highaffinity conformations of interacting cross-sectional layers of the six helices. As a result, this structure is unusually stable, with an apparent melting temperature of 78 °C in the presence of the denaturant guanidine hydrochloride at 5 M concentration. The extended HR regions may therefore be required to prime the group 1 S glycoproteins for their fusion-activating conformational changes during viral entry. Our results provide an initial basis for understanding an intriguing interplay between the presence or absence of proteolytic maturation among the coronavirus groups and the membrane fusion activity of their S glycoproteins. This study also suggests a potential strategy for the development of improved HCoVNL63 fusion inhibitors.

Liu, J, Deng, Y, Zheng, Q, Cheng, CS, Kallenbach, NR, Lu M. A Parallel Coiled-Coil Tetramer with Offset Helices. Biochemistry 45: 15224-15231

Specific helix-helix interactions are fundamental in assembling the native state of proteins and in protein-protein interfaces. Coiled coils afford a unique model system for elucidating principles of molecular recognition between R helices. The coiled-coil fold is specified by a characteristic seven amino acid repeat containing hydrophobic residues at the first (a) and fourth (d) positions. Nonpolar side chains spaced three and four residues apart are referred to as the 3-4 hydrophobic repeat. The presence of apolar amino acids at the e or g positions (corresponding to a 3-3-1 hydrophobic repeat) can provide new possibilities for close-packing of R-helices that includes examples such as the lac repressor tetramerization domain. Here we demonstrate that an unprecedented coiled-coil interface results from replacement of three charged residues at the e positions in the dimeric GCN4 leucine zipper by nonpolar valine side chains. Equilibrium circular dichroism and analytical ultracentrifugation studies indicate that the valinecontaining mutant forms a discrete R-helical tetramer with a significantly higher stability than the parent leucine-zipper molecule. The 1.35 Å resolution crystal structure of the tetramer reveals a parallel fourstranded coiled coil with a three-residue interhelical offset. The local packing geometry of the three hydrophobic positions in the tetramer conformation is completely different from that seen in classical tetrameric structures yet bears resemblance to that in three-stranded coiled coils. These studies demonstrate that distinct van der Waals interactions beyond the a and d side chains can generate a diverse set of helix-helix interfaces and three-dimensional supercoil structures.

Forouhar F, Anderson JL, Mowat CG, Vorobiev SM, Hussain A, Abashidze M, Bruckmann C, Thackray SJ, Seetharaman J, Tucker T, Xiao R, Ma LC, Zhao L, Acton TB, Montelione GT, Chapman SK, Tong L. 'Molecular insights into substrate recognition and catalysis by tryptophan 2,3-dioxygenase.' Proc Natl Acad Sci U S A 104: 473-478

Tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) constitute an important, yet relatively poorly understood, family of heme-containing enzymes. Here, we report extensive structural and biochemical studies of the _Xanthomonas campestris_ TDO and a related protein SO4414 from _Shewanella oneidensis_, including the structure at 1.6-Å resolution of the catalytically active, ferrous form of TDO in a binary complex with the substrate L-Trp. The carboxylate and ammonium moieties of tryptophan are recognized by electrostatic and hydrogen-bonding interactions with the enzyme and a propionate group of the heme, thus defining the L-stereospecificity. A second, possibly allosteric, L-Trp-binding site is present at the tetramer interface. The sixth coordination site of the heme-iron is vacant, providing a dioxygenbinding site that would also involve interactions with the ammonium moiety of L-Trp and the amide nitrogen of a glycine residue. The indole ring is positioned correctly for oxygenation at the C2 and C3 atoms. The active site is fully formed only in the binary complex, and biochemical experiments confirm this induced-fit behavior of the enzyme. The active site is completely devoid of water during catalysis, which is supported by our electrochemical studies showing significant stabilization of the enzyme upon substrate binding.

• Nov 2006

Xiang S, Usunow G, Lange G, Busch M, Tong L 'Crystal structure of 1-deoxy-D-xylulose 5-phosphate synthase (DXS), a crucial enzyme for isoprenoids biosynthesis.' J Biol Chem 282: 2676-2682

Isopentenyl pyrophosphate (IPP) is a common precursor for the synthesis of all isoprenoids, which have important functions in living organisms. IPP is produced by the mevalonate pathway in archaea, fungi and animals. In contrast, IPP is synthesized by a mevalonateindependent pathway in most bacteria, algae, and plant plastids. 1-deoxy-Dxylulose 5-phosphate synthase (DXS) catalyzes the first and the rate-limiting step of the mevalonate-independent pathway, and is an attractive target for the development of novel antibiotics, antimalarials, and herbicides. We report here the first structural information on DXS, from E. coli and D. radiodurans, in complex with the coenzyme thiamine pyrophosphate (TPP). The structure contains three domains (I, II and III), each of which bears homology to the equivalent domains in transketolase and the E1 subunit of pyruvate dehydrogenase. However, DXS has a novel arrangement of these domains as compared to the other enzymes, such that the active site of DXS is located at the interface of domains I and II in the same monomer, whereas that of transketolase is located at the interface of the dimer. The coenzyme TPP is mostly buried in the complex, but the C2 atom of its thiazolium ring is exposed to a pocket that is the substrate binding site. The structures identify residues that may have important roles in catalysis, which have been confirmed by our mutagenesis studies.

Mandel CR, Kaneko S, Zhang H, Gebauer D, Vethantham V, Manley JL, Tong L 'Polyadenylation factor CPSF-73 is the pre-mRNA 3'-end-processing endonuclease.' Nature 282: 953-956

Most eukaryotic messenger RNA precursors (pre-mRNAs) undergo extensive maturational processing, including cleavage and polyadenylation at the 3’-end^1-8. Despite the characterization of many proteins that are required for the cleavage reaction, the identity of the endonuclease is not known^4,9,10 Recent analyses indicated that the 73-kDa subunit of cleavage and polyadenylation specificity factor (CPSF-73) might be the endonuclease for this and related reactions^10-15, although no direct data confirmed this. Here we report the crystal structures of human CPSF-73 at 2.1 Å resolution, complexed with zinc ions and a sulphate that might mimic the phosphate group of the substrate, and the related yeast protein CPSF-100 (Ydh1) at 2.5 Å resolution. Both CPSF-73 and CPSF-100 contain two domains, a metallo-b-lactamase domain and a novel β-CASP (named for metallo-β-lactamase, CPSF, Artemis, Snm1, Pso2) domain¹². The active site of CPSF-73, with two zinc ions, is located at the interface of the two domains. Purified recombinant CPSF-73 possesses RNA endonuclease activity, and mutations that disrupt zinc binding in the active site abolish this activity. Our studies provide the first direct experimental evidence that CPSF- 73 is the pre-mRNA 3’-end-processing endonuclease.

Lemaster DM, Hernandez G 'Residue cluster additivity of thermodynamic stability in the hydrophobic core of mesophile vs. hyperthermophile rubredoxins.' Biophys Chem 125: 483-489

Transient conformational dynamics have long been inferred to play a critical role in the biological functions of proteins. Recently, more direct evidence for the role of conformational fluctuations in catalysis has been found for enzymes which undergo concerted transitions in the active site that occur within the timeframe of substrate turnover.^[1–4] Mutations which disrupt these collective active site transitions result in reduced catalysis.^{[5, 6]} Monitoring the internal mobility of residues that are distant from the mutation site can provide insight into how conformational dynamics propagate through the protein structure. However, in such studies the evidence of substantial changes in dynamics at distal sites have generally arisen from mutations that produce a significant decrease in global stability.^{[ 7–10]} The disruption of native-like interactions that result from such destabilizing mutations complicate the interpretation of the differential conformational dynamics in terms of motions that reflect the parental protein structure.

• Oct 2006

Liu J, Zheng Q, Deng Y, Cheng CS, Kallenbach NR, Lu M 'A seven-helix coiled coil.' Proc Natl Acad Sci U S A 103: 15457–15462

Coiled-coil proteins contain a characteristic seven-residue sequence repeat whose positions are designated a to g. The interacting surface between α-helices in a classical coiled coil is formed by interspersing nonpolar side chains at the a and d positions with hydrophilic residues at the flanking e and g positions. To explore how the chemical nature of these core amino acids dictates the overall coiled-coil architecture, we replaced all eight e and g residues in the GCN4 leucine zipper with nonpolar alanine side chains. Surprisingly, the alanine-containing mutant forms a stable α-helical heptamer in aqueous solution. The 1.25-Å resolution crystal structure of the heptamer reveals a parallel seven-stranded coiled coil enclosing a large tubular channel with an unusual heptad register shift between adjacent staggered helices. The overall geometry comprises two interleaved hydrophobic helical screws of interacting cross-sectional a and d layers that have not been seen before. Moreover, asparagines at the a positions play an essential role in heptamer formation by participating in a set of buried interhelix hydrogen bonds. These results demonstrate that heptad repeats containing four hydrophobic positions can direct assembly of complex, higher-order coiled-coil structures with rich diversity for close packing of α-helices.

Armstrong N, Jasti J, Beich-Frandsen M, Gouaux E 'Measurement of Conformational Changes accompanying Desensitization in an Ionotropic Glutamate Receptor.' Cell 127: 85-97

The canonical conformational states occupied by most ligand-gated ion channels, and many cell-surface receptors, are the resting, activated, and desensitized states. While the resting and activated states of multiple receptors are well characterized, elaboration of the structural properties of the desensitized state, a state that is by definition inactive, has proven difficult. Here we use electrical, chemical, and crystallographic experiments on the AMPAsensitive GluR2 receptor, defining the conformational rearrangements of the agonist binding cores that occur upon desensitization of this ligand- gated ion channel. These studies demonstrate that desensitization involves the rupture of an extensive interface between domain 1 of 2-fold related glutamate-binding core subunits, compensating for the ca. 21° of domain closure induced by glutamate binding. The rupture of the domain 1 interface allows the ion channel to close and thereby provides a simple explanation to the long-standing question of how agonist binding is decoupled from ion channel gating upon receptor desensitization.

Mandel CR, Gebauer D, Zhang H, Tong L 'A serendipitous discovery that in situ proteolysis is essential for the crystallization of yeast CPSF-100 (Ydh1p).' Acta Crystallograph Sect F Struct Biol Cryst Commun 62: 1041-5

The cleavage and polyadenylation specificity factor (CPSF) complex is required for the cleavage and polyadenylation of the 3'-end of messenger RNA precursors in eukaryotes. During structural studies of the 100 kDa subunit (CPSF-100, Ydh1p) of the yeast CPSF complex, it was serendipitously discovered that a solution that is infected by a fungus (subsequently identified as Penicillium) is crucial for the crystallization of this protein. Further analyses suggest that the protein has undergone partial proteolysis during crystallization, resulting in the deletion of an internal segment of about 200 highly charged and hydrophilic residues, very likely catalyzed by a protease secreted by the fungus. With the removal of this segment, yeast CPSF-100 (Ydh1p) has greatly reduced solubility and can be crystallized in the presence of a minute amount of precipitant.

• Sep 2006

Stiegler AL, Burden SJ, Hubbard SR 'Crystal Structure of the Agrin-responsive immunoglobulin-like Domains 1 and 2 of the Receptor Tyrosine Kinase MuSK.' J Mol Biol 364: 424-433

Muscle-specific kinase (MuSK) is a receptor tyrosine kinase expressed exclusively in skeletal muscle, where it is required for formation of the neuromuscular junction. MuSK is activated by agrin, a neuron-derived heparan sulfate proteoglycan. Here, we report the crystal structure of the agrin-responsive first and second immunoglobulin-like domains (Ig1 and Ig2) of the MuSK ectodomain at 2.2 Å resolution. The structure reveals that MuSK Ig1 and Ig2 are Ig-like domains of the I-set subfamily, which are configured in a linear, semi-rigid arrangement. In addition to the canonical internal disulfide bridge, Ig1 contains a second, solvent-exposed disulfide bridge, which our biochemical data indicate is critical for proper folding of Ig1 and processing of MuSK. Two Ig1-2 molecules form a non-crystallographic dimer that is mediated by a unique hydrophobic patch on the surface of Ig1. Biochemical analyses of MuSK mutants introduced into MuSK−/− myotubes demonstrate that residues in this hydrophobic patch are critical for agrin-induced MuSK activation.

Bennett MT, Rodgers MT, Hebert AS, Ruslander LE, Eisele L, Drohat AC 'Specificity of Human Thymine DNA Glycosylase Depends on N-Glycosidic Bond Stability.' J Am Chem Soc 128: 12510-12519

Initiating the DNA base excision repair pathway, DNA glycosylases Trash.findDFdf and hydrolytically excise damaged bases from DNA. While some DNA glycosylases exhibit narrow specificity, others remove multiple forms of damage. Human thymine DNA glycosylase (hTDG) cleaves thymine from mutagenic G‚T mispairs, recognizes many additional lesions, and has a strong preference for nucleobases paired with guanine rather than adenine. Yet, hTDG avoids cytosine, despite the million-fold excess of normal G‚C pairs over G‚T mispairs. The mechanism of this remarkable and essential specificity has remained obscure. Here, we examine the possibility that hTDG specificity depends on the stability of the scissile base-sugar bond by determining the maximal activity ( k_max) against a series of nucleobases with varying leaving-group ability. We Trash.findDFdf that hTDG removes 5-fluorouracil 78-fold faster than uracil, and 5-chlorouracil, 572-fold faster than thymine, differences that can be attributed predominantly to leaving-group ability. Moreover, hTDG readily excises cytosine analogues with improved leaving ability, including 5-fluorocytosine, 5-bromocytosine, and 5-hydroxycytosine, indicating that cytosine has access to the active site. A plot of log( k_max) versus leaving-group p K _a reveals a Brønsted-type linear free energy relationship with a large negative slope of β_lg )-1.6 ( 0.2, consistent with a highly dissociative reaction mechanism. Further, we Trash.findDFdf that the hydrophobic active site of hTDG contributes to its specificity by enhancing the inherent differences in substrate reactivity. Thus, hTDG specificity depends on N-glycosidic bond stability, and the discrimination against cytosine is due largely to its very poor leaving ability rather than its exclusion from the active site.

Natarajan, A., Ghose, R., M. Hill, J., Structure and Dynamics of ASC2, A Pyrin Domain-Only Protein that Regulates Inflammatory Signalling. J Biol Chem. 281: 31863-31875

Pyrin domain (PYD)-containing proteins are key components of pathways that regulate inflammation, apoptosis, and cytokine processing. Their importance is further evidenced by the consequences of mutations in these proteins that give rise to autoimmune and hyperinflammatory syndromes. PYDs, like other members of the death domain (DD) superfamily, are postulated to mediate homotypic interactions that assemble and regulate the activity of signaling complexes. However, PYDs are presently the least well characterized of all four DD subfamilies. Here we report the three-dimensional structure and dynamic properties of ASC2, a PYD-only protein that functions as a modulator of multidomain PYD-containing proteins involved in NF-κB and caspase-1 activation. ASC2 adopts a six-helix bundle structure with a prominent loop, comprising 13 amino acid residues, between helices two and three. This loop represents a divergent feature of PYDs from other domains with theDDfold. Detailed analysis of backbone ¹⁵N NMR relaxation data using both the Lipari-Szabo model-free and reduced spectral density function formalisms revealed no evidence of contiguous stretches of polypeptide chain with dramatically increased internal motion, except at the extreme N and C termini. Some mobility in the fast, picosecond to nanosecond timescale, was seen in helix 3 and the preceding α2 - α3 loop, in stark contrast to the complete disorder seen in the corresponding region of the NALP1 PYD. Our results suggest that extensive conformational flexibility in helix 3 and the α2 - α3 loop is not a general feature of pyrin domains. Further, a transition from complete disorder to order of the α2 - α3 loop upon binding, as suggested for NALP1, is unlikely to be a common attribute of pyrin domain interactions.

Del Rio, A., Dutta K., Chavez J., Ubarretxena-Belandia, I., Ghose, R., Solution Structure and Dynamics of the N-terminal Cytosolic Domain of Rhomboid Intramembrane Protease from Pseudomonas aeruginosa: Insights into a Functional Role in Intramembrane Proteolysis. J Mol Biol 365: 109-122

Rhomboids are ubiquitous integral membrane proteases that release cellular signals from membrane-bound substrates through a general signal transduction mechanism known as regulated intramembrane proteolysis (RIP). We present the NMR structure of the cytosolic N-terminal domain (NRho) of P. aeruginosa Rhomboid. NRho consists of a novel α/β fold and represents the first detailed structural insight into this class of intramembrane proteases. We Trash.findDFdf evidence that NRho is capable of strong and specific association with detergent micelles that mimic the membrane/ water interface. Relaxation measurements on NRho reveal structural fluctuations on the microseconds–milliseconds timescale in regions including and contiguous to those implicated in membrane interaction. This structural plasticity may facilitate the ability of NRho to recognize and associate with membranes. We suggest that NRho plays a role in scissile peptide bond selectivity by optimally positioning the Rhomboid active site relative to the membrane plane.

Massi F, Wang C, Palmer AG. Solution NMR and computer simulation studies of active site loop motion in triosephosphate isomerase. Biochemistry 36: 10787-94

Solution NMR spin relaxation experiments and classical MD simulations are used to study the dynamics of triosephosphate isomerase (TIM) in complex with glycerol 3-phosphate (G3P). Three regions in TIM exhibit conformational transitions on the μs-ms time scale as detected by chemical exchange broadening effects in NMR spectroscopy: residue Lys 84 on helix C, located at the dimeric interface; active site loop 6; and helix G. The results indicate that the conformational exchange process affecting the residues of loop 6 is the correlated opening and closing of the loop. Distinct processes are responsible for the chemical exchange linebroadening observed in the other regions of TIM. MD simulations confirm that motions of individual residues within the active site loop are correlated and suggest that the chemical exchange processes observed for residues in helix G arise from transitions between 3₁₀- and α-helical structures. The results of the joint NMR and MD study provide global insight into the role of conformational dynamic processes in the function of TIM.

"Protein backbone dynamics through 13C´ -13Cα cross-relaxation in NMR spectroscopy. " Fabien Ferrage , Philippe Pelupessy , David Cowburn, Geoffrey Bodenhausen., J. Am. Chem. Soc. 128: 11072-8

Internal dynamics of proteins are usually characterized by the analysis of 15N relaxation rates that reflect the motions of NHN vectors. It was suggested a decade ago that additional information on backbone motions can be obtained by measuring cross-relaxation rates associated with intra-residue C¢CR vectors. Here we propose a new approach to such measurements, based on the observation of the transfer between two-spin orders 2NzC¢z and 2NzCz R . This amounts to “anchoring” the C¢z and Cz R operators to the Nz term from the amide of the next residue. In combination with symmetrical reconversion, this method greatly reduces various artifacts. The experiment is carried out on human ubiquitin at 284.1 K, where the correlation time is 7.1 ns. The motions of the C¢CR vector appear more restricted than those of the NHN vector.

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