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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.
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.
| 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 |  |
Trigger factor (TF) is a eubacterial chaperone that associates with ribosomes at the peptide-exit tunnel and also occurs in excess free in the cytosol. TF is a three-domain protein that appears to exist in a dynamic equilibrium of oligomerization states and interdomain conformations. X-ray crystallography and chemical cross-linking were used to study the roles of the N- and C-terminal domains of Thermotoga maritima TF in TF oligomerization and chaperone activity. The structural conservation of both the N- and C-terminal TF domains was unambiguously established. The biochemical and crystallographic data reveal a tendency for these domains to partake in diverse and apparently nonspecific protein–protein interactions. It is found that the T. maritima and Escherichia coli TF surfaces lack evident exposed hydrophobic patches. Taken together, these data suggest that TF chaperones could interact with nascent proteins via hydrophilic surfaces.
| 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 | |
We report here the crystal structure at 2.0 Å resolution of the AGR_C_4470p protein from the Gramnegative
bacterium Agrobacterium tumefaciens. The protein is a tightly associated dimer, each subunit
of which bears strong structural homology with the two domains of the heme utilization protein ChuS
from Escherichia coli and HemS from Yersinia enterocolitica. Remarkably, the organization of the
AGR_C_4470p dimer is the same as that of the two domains in ChuS and HemS, providing structural
evidence that these two proteins evolved by gene duplication. However, the binding site for heme, while
conserved in HemS and ChuS, is not conserved in AGR_C_4470p, suggesting that it probably has a
different function. This is supported by the presence of two homologs of AGR_C_4470p in E. coli, in
addition to the ChuS protein.
| 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.
| 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 detergent1,2 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
| 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 | |
Sec1/Munc18 (SM) proteins are required for every
step of intracellular membrane fusion, but
their molecular mechanism of action has been
unclear. In this work, we demonstrate a fundamental
role of the SM protein: to act as a stimulatory
subunit of its cognate SNARE fusion
machinery. In a reconstituted system, mammalian
SNARE pairs assemble between bilayers to
drive a basal fusion reaction. Munc18-1/nSec1,
a synaptic SM protein required for neurotransmitter
release, strongly accelerates this reaction
through direct contact with both t- and
v-SNAREs. Munc18-1 accelerates fusion only
for the cognate SNAREs for exocytosis, therefore
enhancing fusion specificity.
| 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.
| 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’-end1-8. Despite the characterization
of many proteins that are required for the cleavage reaction, the
identity of the endonuclease is not known4,9,10 Recent analyses
indicated that the 73-kDa subunit of cleavage and polyadenylation
specificity factor (CPSF-73) might be the endonuclease for this and
related reactions10-15, 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.
| 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.
| 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 | |
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