We have solved the crystal structure of the influenza A virus NP to 3.15 Å resolution using multiple isomorphous replacement (MIR). The NP crystals belong to the space group C2221, with a trimer per asymmetric unit. The overall shape of a NP monomer resembles a fetus with a head and a body domain.  A flexibly-attached tail loop interacts with a neighbouring NP and mediates homo-oligomerization in NP oligomers and presumably also in RNPs.  Between the NP head and body domain is a deep, positively charged groove that is likely to function as the RNA binding site.  The viral RNA may wrap through the groove of a NP molecule and then continues to the neighboring subunit, consistent with our biochemical data that each NP binds to a ~20-nucleotide long RNA.  The structure of NP also suggests possible mechanisms for NP interactions with  cellular macromolecules and the viral polymerase. Our studies have significantly enhanced our understanding of NP’s biological function, and should provide promising leads for new anti-influenza drug development.

Polyadenylation of mRNAs in pox viruses is carried out by a poly(A) polymerase heterodimer comprised of a catalytic component, VP55, and a processivity factor, VP39. We have solved the crystal structures of the ATP-γ-S-bound and unbound vaccinia poly(A) polymerase. VP55, which  shows an unusual architecture, is composed of a N-terminal, central or catalytic and C-terminal domains that differ from many polymerases.  Residues in the active site of VP55, which is located between the catalytic and C-terminal domains, make specific interactions with the adenine of the ATP analog, establishing the molecular basis of ATP recognition. The concave surface of  VP55 docks the globular VP39 which makes contacts to each of the three domains of VP55. The heterodimer interface forms a groove that can partially enclose the mRNA substrate, preventing its dissociation from the heterodimer.  A model of RNA binding to the heterodimer, based on biochemical and structural data, is presented.

Previous crystal structures of AK have implicated two important catalytic residues.  The first residue, asp318, looks to be a general base in the transfer of the g-phosphate to the adenosine 5'-OH. The second residue, arg136, stabilizes the in-flight g-phosphate. Mutations of these residues results in a substantial decrease in the kcat with smaller changes in Km. The alanine parameters seem anomalously robust, and were equivalent to that of the glutamate mutant.  Possible scenarios for this activity would be the presence of buffer or water that might function as the general base. In order to understand the structure-function of these residues, work has been directed towards the crystallization of these mutants.  Crystal data for the ternary structures of the mutants have been collected and preliminarily analyzed.
      AK has been characterized from seven sources and there is no
consensus on the kinetic mechanism.  Most AK's have been characterized as ordered Bi-Bi mechanisms, though there is disagreement as to whether the adenosine or ATP binds first and which product is released first.  Evidence
also exists for a two-site ping-pong mechanism involving formation of a
phosphorylenzyme intermediate.  A previous ternary structure of AK with
AMP-PCP and adenosine suggests a direct transfer of the g-phosphate and dramatic domain movements upon substrate binding.  Adenosine makes multiple contacts with both domains in the newly formed active site. The structure of AK bound to two adenosines has also been previously solved and demonstrates the same domain movements, suggesting that adenosine plays a large role in the triggering this movement.  To complete the story apo-AK of WT, D318E, and D318A was soaked with AMP-PCP and the structure solved. The resulting structures are very similar to that of the apo structure.

 

+Department of Biochemistry and Molecular Biology, University of Texas Health
Sciences Center Houston, Houston, TX 77225


Calmodulin binds to a conserved site in ryanodine receptors to regulate Ca2+
release.  Overlapping peptides corresponding to this target induce collapse of
CaM to different extents.  The crystal structure of Ca2+-calmodulin bound to a
30-residue peptide reveals that hydrophobic anchor residues in the target
arranged in a novel '1-17' spacing allow each calmodulin lobe to interact with
the peptide independently.  Solution NMR residual dipolar couplings confirm
the structure of each calmodulin lobe but show that the relative orientation
of the lobes is not fixed.  The independence of the two lobes of calmodulin in
the complex suggests a structural basis for how other domains may compete for
binding to this region to regulate the channel.  Structure comparison reveals
that residues flanking the putative hydrophobic anchors of a calmodulin target
sequence can favor binding in a '1-14' or '1-17' manner. 

 

Pyruvate dehydrogenase kinase isoforms (PDK1- 4) are the molecular switch that down-regulates activity of the human pyruvate dehydrogenase complex (PDC) through reversible phosphorylation. We showed previously that binding of the lipoyl domain 2 (L2) of PDC to PDK3 induces a “cross-tail” conformation in PDK3, resulting in an opening of the active-site cleft and the stimulation of kinase activity. In the present study, we report that alanine-scanning substitutions of L140, E170 and E179 from L2 markedly reduce binding affinities of these L2 mutants for PDK3. Unlike wild-type L2, binding of these L2 mutants to PDK3 does not preferentially reduce the affinity of PDK3 for ADP over ATP. The inefficient removal of product inhibition associated with ADP accounts for the decreased stimulation of PDK3 activity by these L2 variants. Serial truncations of the PDK3 C-terminal tail region also impede or abolish the binding of wild-type L2 to the PDK3 mutants, resulting in the reduction or absence of L2-inhanced kinase activity. Alanine substitutions of residues L27, F32, F35 and F48 in the lipoyl-binding pocket of PDK3 similarly nullify L2 binding and L2-stimulated PDK3 activity. Our results indicate that the above residues in L2 and residues in the C-terminal region and the lipoyl-binding pocket of PDK3 are critical determinants for the interactions between L2 and PDK3, which impart the augmentation of kinase activity.

¹Department of Biochemistry and Molecular Biology, M.D. Anderson
Cancer Center, Houston, TX, 77030, USA.
²School of Biological Sciences, A12, University of Sydney, Sydney NSW
2006, Australia

The repressor QacR regulates the expression of the multidrug exporter
QacA, which mediates antiseptic resistance of Staphylococcus aureus to
a wide variety of molecules.  The ligands of both of QacA and QacR are
lipophilic cations.  In crystal structures of wild-type-QacR-ligand
complexes, glutamates 90 and 120 appear to be critical for binding
rhodamine-6G, ethidium, malachite green, and dequalinium.  In order to
determine if the negative charge of each of these glutamates plays a
role in ligand binding, these residues were substituted with
glutamines.  The interactions of these substituted proteins with the
structurally characterized QacR ligands: ethidium (Et), malachite
green (MG), dequalinium (Dq), and rhodamine-6G (R6G) were studied.
The affinities of the substituted QacR to each ligand were measured
with tryptophan fluorescence quenching, fluorescence polarization, and
isothermal titration calorimetry.  The structures of the protein
ligand complexes were studied with X-ray crystallography
The structures of the E90Q-QacR-R6G complex, the E90Q-QacR-Et complex,
and the E120Q-QacR-Et complex indicated multiple binding positions
were available to each of the ligands in the substituted proteins
compared to the one binding position observed in the WT-QacR.  As the
binding affinity of the substituted proteins for these ligands did not
dramatically change from that of the WT, these new binding positions
may be accessible in the WT-QacR.  This research further demonstrates
how the QacR binding pocket binds a multitude of disparately shaped
ligands.  Not only does this research confirm that the binding pocket
changes to accommodate various ligands, but it illustrates that the
same ligand can interact with the protein in more than one way.

X-ray crystallography is the most powerful tool allowing structural biologists to discern protein structures with the highest degree of detail possible in three dimensions.  During the past few years with the era of structural genomics arriving, much progress has been made in developing high throughput technologies for protein cloning, expression, purification, crystallization, crystal imaging, in house X-ray system and synchrotron beamline data collection.  These new technologies fundamentally impact the approaches of structural biology, the targets at which we can aim as well as the cost and speed of structure determination.  In this report, we reveal the series of new technologies and their apparatuses used from crystallizing proteins to generating electron density maps.  These technologies and apparatuses allow significantly smaller amounts of materials to be used at all steps, along with speedy data collection and accurate phase determination without derivatization.  Ultimately, these open the new paths from gene to structure.

The JCSG is funded by the Protein Structure Initiative of the National Institutes of Health, National Institute of General Medical Sciences. SSRL operations is funded by DOE BES, and the SSRL Structural Molecular Biology program by DOE BER, NIH NCRR BTP and NIH NIGMS.

Data will also be presented from studies demonstrating the use of microfluidic liquid diffusion-based crystallization in a more traditional crystallization pipeline. Follow-on translation strategies from initial screening hits will also be described. Data will be presented from projects which have led to the successful determination of structures from TOPAZ® screening hits.

 Recent trends in macromolecular crystallography have dictated the development of methods that facilitate high-throughput experiments, specifically at synchrotron sources. Equally as important but often less emphasized in the success of these experiments are the procedures that take place in the home laboratory. From crystallization to characterization, advances in technology have not only  improved high-throughput applications but also extended capabilities at home. This presentation will focus on recent developments in Bruker AXS hardware that are key to high-throughput experiments in the home laboratory.

Mutations to the human DNA helicase, hRecQL4 or RTS, give rise to genomic instability that in turn is responsible for a group of autosomal recessive conditions including Rothmund-Thomson Syndrome (RTS) and RAPADILINO. A variety of symptoms is associated with the disorder such as poikiloderma, juvenile cataracts, photosensitivity, skeletal dysplasias and a predisposition to the development of osteosarcomas which develop within the first three to six months of life. The symptoms and severity of the syndrome are correlated to mutations in RecQL4 whose effects are poorly understood. hRecQL4 is currently classified as a RecQ family helicase. This family of proteins includes the Werner’s (WRN) and Bloom’s (BLM) Syndrome helicases which function during DNA repair and replication restart. Recent work from our lab and that of our collaborators (A. Venkitaraman, Cambridge University) suggest that although there are some similarities to WRN and BLM, hRecQL4 plays an essential role in the initiation of DNA replication and has a quaternary structure unlike WRN and BLM. These data together with the unique clinical phenotypes associated with mutation of hRecQL4 suggest a distinct biochemical function for hRecQL4. We are using a combination of structural and biophysical techniques to determine the structural basis for the function of hRecQL4 and the specific defects caused by its mutation.

§Rigaku, 9009 New Trails Dr., The Woodlands, TX 77381-5209 / Tel: +1 (281)
363-1033 / Fax: +1 (281) 364-3628 / www.Rigaku.com
‡Proteros Biostructures GmbH, Am Klopferspitz 19, D-82152
Planegg-Martinsried, Germany / Tel: +49 (0) 89 700761 -0 / www.proteros.de


Protein crystal optimization can be achieved by manipulating a number of
parameters, including: salt or buffer concentration, addition of PEG,
dehydration in air or under oil, and flash annealing. These classical
approaches, aimed at changing the water content in the crystals, often
suffer from irreproducibility and the inability for some crystals to
tolerate the environmental extremes typical of these experiments. A novel
tool, the Proteros Free Mounting System (FMS), allows for accurate control
of protein crystal water content, which can greatly improve diffraction
quality of difficult crystals. The FMS precisely controls the relative
humidity of the gas stream enveloping the crystal while the X-ray
diffraction pattern is monitored in real time, providing direct feedback.
Results show that many protein crystals can be optimized in terms of
resolution, mosaicity or anisotropy. Though the degree of optimization
varies from one protein crystal system to another, the FMS provides an
important tool for flash-cooling crystals when addition of cryoprotectants
causes a loss in diffraction quality. Further, these results are
reproducible and sometimes reversible, allowing for optimization and data
collection from a single crystal.
 

Although dsRNA viruses, birnaviruses possess characteristic features of +ssRNA viruses in genome organization, replication strategy, polyprotein coding strategy and capsid fold. The novel polymerase active site topology, which is now observed in VP1 crystal structure, may also be adopted by polymerases from Thosea asigna virus (TaV) and Euprosterna elaeasa virus (EeV) in the alphavirus superfamily based on primary sequence homology. Therefore, the VP1 structure lends further support to the notion that birnaviruses are closely related to +ssRNA viruses, and they may have stemmed from a common ancestor as TaV/EeV after the polymerase permutation has occurred, revealing the evolutionary relationship between dsRNA and +ssRNA viruses.

Analysis of the Spx aCTD complex structure provides a mechanism for repression of activator stimulated transcription by Spx, but the mechanism of transcription activation by the Spx aCTD complex remains less clear.   However, comparison of the Spx structure to other transcription activators provides some insight into the global regulatory mechanism of Spx.

IL-24 contains three putative N-linked glycosylation sites and two cysteines that, according to our homology model, are close enough to form a unique disulphide bridge.  We have removed both the N-glycosylation sites and potential disulphide bridge using site directed mutagenesis.  Our preliminary data suggests that while the glycosylation sites are occupied, disulphide linkage cannot be confirmed.  Deletion of N-linked glycosylation or disulphide potential does not negate activity.  Unlike the other homologous family members (IL-10, IL-19, IL-22, IL-20, IL-26), deletion of the disulphide linkage may not disrupt biological activity.