B8. Applications of X-ray crystallography in life sciences Lectures

B8. Applications of X-ray crystallography in life sciences
Lectures
L14.2
L14.1
Structural studies of proteins
from silkworm hemolymph
The link between CG methylation and
depletion across the kingdoms of life
Marek Wojciechowski, Honorata
Czapinska, Matthias Bochtler
International Institute of Molecular and Cell Biology, Warsaw, Poland
e-mail: Matthias Bochtler <[email protected]>
DNA methylation occurs in prokaryotes and eukaryotes,
but in different forms and with different functions. In
prokaryotes, methylation is very diverse. Mechanistically,
the modification can affect the N4 or C5 of cytosine or
N6 of adenine. Sequence context is variable. Functionally,
methylation plays a role in restriction modification systems,
in DNA repair for the distinction of parental and daughter
strand, and also in the control of bacterial lifestyle. Some
of this is conserved in primitive eukaryotes, but in higher
eukaryotes, particularly vertebrates, methylation is predominantly reduced to C5 methylation in a single sequence context (CG, more traditionally CpG), and serves to control
the epigenetic state of DNA, in crosstalk with appropriate histone modifications. For eukaryotic organisms with
DNA methylation, it is known that the CG sequence is not
only important, but also rare: in fact, the actual number of
CGs is about fourfold lower than statistically expected. In
my talk, I will discuss the mechanistic link between methylation and the depletion of target sequences and I will address the question about the generality of the link across all
kingdoms of life.
Grzegorz Bujacz1,2, Agnieszka J. Pietrzyk1, Anna
Bujacz2, Santosh Panjikar3, Mariusz Jaskólski1,4
1Center of Biocrystallographic Research, Institute of Bioorganic
Chemistry, Polish Academy of Sciences, Poznan, Poland; 2Technical
University of Lodz, Faculty of Biotechnology and Food Sciences, Insitute
of Technical Biochemistry, Lodz, Poland; 3European Molecular Biology
Laboratory, Hamburg Outstation, Deutsches Elektronen-Synchrotron,
Hamburg, Germany; 4Department of Crystallography, Faculty of
Chemistry, A. Mickiewicz University, Poznan, Poland
e-mail: Grzegorz Bujacz <[email protected]>
Bombyx mori is a one of two species of domesticated insects.
It is a perfect model organism to study insect development.
There are a large number of genes sequenced from this organism, but only a few protein crystal structures have been
determined. The hemolymph is an ideal source of proteins
because it contains a selected subset of all the proteins that
are essential for the insect’s development. We have developed an efficient procedure for the isolation and purification of several major proteins from the hemolymph of the
fifth instar larvae of the mulberry silkworm Bombyx mori.
The proteins represent the two major groups (30 kDa and
80 kDa) found in the hemolymph. The role of a number
of these proteins is still unknown and we hope to elucidate
their function through a structural genomics approach.
We have isolated, purified, and crystallized three proteins
from the first group and collected initial diffraction data for
two of them: Juvenile Hormone Binding Protein (JHBP)
and Bombyx mori lipoprotein 7 Bmlp7. JHBP plays a critical
role in insect metamorphosis and development because it
transports the highly hydrophobic juvenile hormone (JH)
from the site of its synthesis to target tissues. We would like
to study the protein-hormone interactions and elucidate
the role of the second binding pocket of this protein. The
function of Bmlp7 is unknown. The third protein crystals
are small and have poor diffraction properties and require
further optimization.
The crystal structure of Bmlp7 lipoproteins from the mulberry silkworm Bombyx mori L. hemolymph was determined
at 1.3 Å resolution. A unique right-handed super helix is
present in the structure. It is created by six helices of the
N-terminal domain. The crystal structure of Bmlp7 reveals
the homology of the C terminal β-trefoil domain to the
domains of a mosquitocidal toxin from Bacillus sphaericus
and of the main hemagglutinin component of Clostridium
botulinum. The biological role of Bmlp7 is yet unknown.
The homology to the sugar binding proteins might confirm
that this protein can play a role in glucan and other carbohydrate binding, while the central canal of the N-terminal
domain may bind hydrophobic ligands, but this hypothesis
has to be confirmed by crystallizing complexes with such
ligands. Bmlp7 and some other members of the 30 kDa
lipoprotein family have anti-apoptotic properties and are
involved in immune response to fungal infections.
2nd Congress of Biochemistry and Cell Biology, September 5th–9th, 2011, Kraków, Poland
B8. Applications of X-ray crystallography in life sciences
L14.3
L14.4
Monitoring the antagonist-protein and
protein-protein interactions with NMR
spectroscopy and X-ray crystallography
High pressure in structural
studies of macromolecules
Grzegorz M. Popowicz, Michael Bista, Marcin Krajewski,
Loyola D’Silva, Ulli Rotweiller, Mahavir Singh, Tad A. Holak
Max Planck Institute for Biochemistry, Martinsried, Germany
e-mail: Tad A. Holak <[email protected]>
Protein-protein interactions play a pivotal role in virtually every cellular process. Frequently such interactions are
weak and transient. NMR spectroscopy has the unique ability to provide information about such interactions. We will
present a brief overview of new NMR methods for the
characterization of protein-protein interactions. We will
also describe the application of NMR and X-ray crystallography in the identification and characterization of small
molecules for inhibition of protein function — a primary
objective of rational drug design.
A combined application of NMR and X-ray crystallography will be illustrated by studies of the proteins involved
in the p53/retinoblastoma protein (pRb) pathway. The p53
tumor-suppressor gene is dysregulated in human malignancies. The retinoblastoma tumor suppressor protein (pRb) is
a fundamental negative regulator of cell proliferation, and
it is now believed that the p53/pRb pathway is effectively
inactivated in all human cancers.
157
Krzysztof Lewiński, Katarzyna Kurpiewska, Joanna Loch
Faculty of Chemistry, Jagiellonian University, Kraków, Poland
e-mail: Krzysztof Lewiński <[email protected]>
A protein in solution equilibrates among multiple ensembles including native conformer and locally unfolded intermediates that differ in free energy and partial molar volume.
Standard crystal structure reflects an average positions of
atoms and shows the most populated low energy conformers. The application of high pressure shifts equilibrium toward more compact ensembles increasing the population
of higher energy conformers and at some point initiates
unfolding of a protein. Structural investigations by NMR
spectroscopy or X-ray crystallography at elevated pressure
allow us to detect higher energy conformers preceding unfolded states. Increasing number of mostly spectroscopic
high pressure studies of protein folding that is observed
recently is related to observation that some neurodegenerative diseases are associated with formation of misfolded
protein molecules and insoluble protein aggregates. The
mechanism of origination of these proteins has not been
determined yet but it is agreed, that it requires partial unfolding of the protein. Detailed analysis of intramolecular
interactions and factors like mutations that affects them is
important for understanding mechanism of these processes and may help to develop new therapeutic approaches.
The Protein Data Bank contains crystal structures determined at various high pressure conditions for only about
ten proteins. Using diamond anvil cell we collected diffraction data at high pressure conditions and solved the crystal
structure for: RNase wt (670 MPa, 2.35 Å), RNase A I106A
variant (480 MPa, 2.40 Å), insulin (85 MPa, 2.6 Å; 143 MPa,
2.6 Å; 200 MPa, 3.0 Å), β-lactoglobulin (430 MPa, 2.8 Å),
thaumatin (150 MPa, 2.8 Å, 520 MPa, 2.4 Å). Comparison of high pressure structures with reference structures
determined at ambient pressure revealed subtle structural
changes that involve conformations mostly in loop regions
and shift of fragments of molecule. Some of these changes
may be related to initiation of unfolding process.
2nd Congress of Biochemistry and Cell Biology, September 5th–9th, 2011, Kraków, Poland
158
B8. Applications of X-ray crystallography in life sciences
Oral presentations
O14.2
O14.1
Substrate induced activation
of SplB protease studied with
transition state analog inhibitor
The single-point mutation stabilizing the
L1 loop is sufficient to preserve human
cystatin C molecule in its monomeric form
Marta Orlikowska1, Elżbieta Jankowska1, Izabela
Behrendt1, Dominika Borek2, Robert Kołodziejczyk3,
Zbyszek Otwinowski2, Mariusz Jaskólski3,
Piotr Skowron3, Aneta Szymańska1
1Department of Medicinal Chemistry, Faculty of Chemistry,
University of Gdańsk, Gdańsk, Poland; 2Department of Biochemistry,
UT Southwestern Medical Center, Dallas, USA; 3Center for
Biocrystallographic Research, Institute of Bioorganic Chemistry, Polish
Academy of Sciences, Poznań, Poland; 4Division of Environmental
Molecular Biotechnology, Faculty of Chemistry, University of Gdańsk,
Gdańsk, Poland
e-mail: Marta Orlikowska <[email protected]>
Human cystatin C (hCC) is a basic, low molecular mass
protein comprised of 120 amino acid residues (MW 13,343
Da) that belongs to a family of single-chain reversible inhibitors of papain-like (C1 family) and legumain-related
(C13 family) cysteine proteases [1]. Human cystatin C
(hCC) can be found in all tissues and body fluids [2], where
it plays a role of a main regulator of cysteine proteases
activity. Besides its physiological inhibitory function, hCC
is also involved in neuropathological disorders connected
with amyloid deposition. A naturally occurring single-point
mutation in cystatin C gene, changing L68 into Q68, leads
to hereditary cystatin C amyloid angiopathy, Icelandic type
(HCCAA-I), also known as hereditary cerebral hemorrhage
with amyloidosis.
At physiological conditions hCC wt is a monomeric protein, but under crystallization conditions forms the domain
swapped dimer [3]. Loop 1 is the only part of hCC which
undergoes significant structural perturbations during the
dimerization process: its topology changes from a turn to
an extended β-strand. Experimental [4, 5] and theoretical
[6, 7] studies revealed that this region is conformationally
unstable and the main destabilization is connected with Val
residue (for hCC Val57), located on the top of the loop.
Values of the ψ angles for this residue are not optimal what
can account to propensity of the protein to undergo domain swapping.
In order to assess the influence of the unfavourable Val
residue on the hCC molecules, we designed and constructed mutant in which Val57 was substituted by Asp, Asn [8],
Gly (residues favored in this position of β-turns) or Pro, respectively. By applying this rational mutagenesis approach
we were able to preserve human cystatin C molecule in its
monomeric form. Here we report crystal structures of V57
hCC mutants and discuss the architecture of the protein in
comparison to the native protein.
Michal Zdzalik1, Ewa Pietrusewicz3, Przemyslaw Golik1,
Justyna Stec-Niemczyk1, Benedykt Wladyka2, Jan
Potempa1, Jozef Oleksyszyn3, Grzegorz Dubin1
1Department of Microbiology and 2Department of Analytical
Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology,
Jagiellonian University, Krakow, Poland; 3Division of Medicinal
Chemistry and Microbiology, Faculty of Chemistry, Wroclaw University
of Technology, Wroclaw, Poland
e-mail: Michal Zdzalik <[email protected]>
Staphylococcus aureus is a widespread opportunistic human
pathogen causing a wide range of diseases. Among staphylococcal virulence factors extracellular proteases are
counted. This study focuses on SplB protease, the member of a recently described group of six serine proteases
(SplA-F) expressed from a single operon by multiple staphylococcal strains. To date crystal structure of SplB in a
native form was solved revealing an interesting structural
deformation of the catalytic site of the enzyme. The deformation should in theory preclude the activity of the
otherwise demonstrated active enzyme. The structure referred to as the oxyanion hole is present at the catalytic site
of serine proteases where it is necessary for stabilization
of reaction intermediate. Analysis of native SplB crystal
structure revealed no preformed oxyanion hole. It was thus
unclear whether the enzyme was active despite the lack of
preformed oxyanion hole or the protease is activated upon
specific substrate binding. To solve the ambiguity two crystal structures of SplB complexed with substrate mimicking
phosphonate inhibitor were solved. Crystallographic models were obtained 1.7 Å and 1.9 Å resolution in C2 and
P212121 space groups, respectively. The structures reveal
the presence of phosphonate oxygen of the inhibitor in
the fully formed oxyanion hole of SplB protease. The arrangement of the oxyanion hole forming residues in the
inhibitor bound structures of SplB is similar to that found
in canonical S1 family proteases. This finding demonstrates
that SplB protease is activated upon substrate binding by
induced rearrangement of residues forming the oxyanion
hole.
References
Turk V, Bode W (1991) FEBS Lett 285: 213-219.
Grubb A (200) Adv Clin Chem 35: 63-99.
Janowski R et al. (2001) Nat Struct Biol 8: 316-320.
Engh R et al. (1993) J Mol Biol 234: 1060-1069.
Martin J et al. (1995) J Mol Biol 246: 331-343.
Ding F et al. (2006) Structure 14: 5-14.
Rodziewicz-Motowidło S et al. (2009) Biopolymers 91: 373-83.
Orlikowska M et al. (2011) J Struct Biol 173: 406-413 .
2nd Congress of Biochemistry and Cell Biology, September 5th–9th, 2011, Kraków, Poland
B8. Applications of X-ray crystallography in life sciences
Posters
P14.2
P14.1
Synthesis and study of steric zipper motif
in the sequence of human cystatin C
Large-scale screening for crystallization
conditions of major hemolymph proteins
from mulberry silkworm Bombyx mori L.
Agnieszka J. Pietrzyk1, Jochen Mueller-Dieckmann2,
Mariusz Jaskólski1,3, Grzegorz Bujacz1,4
1Polish
Academy of Sciences, Institute of Bioorganic Chemistry, Center
of Biocrystallographic Research, Poznan, Poland; 2European Molecular
Biology Laboratory, Hamburg Outstation, Deutsches ElektronenSynchrotron, Hamburg, Germany; 3A. Mickiewicz University, Faculty of
Chemistry, Department of Crystallography, Poznan, Poland; 4Technical
University of Lodz, Faculty of Biotechnology and Food Sciences, Insitute
of Technical Biochemistry, Lodz, Poland
e-mail: Agnieszka Pietrzyk <[email protected]>
Mulberry silkworm Bombyx mori L. is the main producer of
natural silk but it is not the only aspect that makes studies of this insect important. For instance, the silkworm
hemolymph has antiapoptotic properties. The addition of
hemolymph to HEK and HeLa cell cultures inhibits the
apoptosis of the cells (Rhee et al., 2007). The components
involved in this inhibition are major hemolymph proteins,
which are specific to the final instar larvae stage of mulberry silkworm. The proteins could be divided into two main
fractions, the high molecular weight and the low molecular
weight fraction. The latter one consists mainly of 30 kDa
lipoproteins and juvenile hormone binding protein (BmJHBP). In contrast to 30 kDa lipoproteins, BmJHBP is not
involved in apoptosis inhibition but this juvenile hormone
carrier is one of the most important proteins for silkworm
development.
The high molecular weight and low molecular weight fractions of silkworm hemolymph have been separated using gel filtration on Superdex 200 pg resin. Ion exchange
chromatography was carried out as the next purification
step to isolate homogeneous proteins from both fractions.
According to this protocol, BmJHBP and two unknown
proteins (labeled BmX and BmY) belonging to the 30 kDa
lipoprotein family were purified from the low molecular weight fraction. Three other proteins (labeled BmA1,
BmA2 and BmA4) of higher molecular weight were also
isolated. Their molecular mass has been determined by
MALDI-TOF MS analysis to be about 80 kDa. Searching
for crystallization conditions was carried out using highthroughput crystallization methodology and commercially
available screening buffers. So far, initial crystallization
conditions have been found for one of the high molecular weight proteins (BmA4). Crystallization conditions for
all of the isolated low molecular weight proteins could be
found as well, but in the case of BmJHBP and BmY the
quality of the crystals is not satisfactory. The crystals of
BmX were of a good quality and diffracted X-rays to 1.3
A resolution, enabling us to solve the protein structure and
identify the correct amino acid sequence.
159
Emilia Górna1, Natalia Maciejewska1, Mariusz
Jaskólski2, Aneta Szymańska1
1Department of Medicinal Chemistry, University of Gdańsk, Gdańsk,
Poland; 2Department of Crystallography, Faculty of Chemistry,
A. Mickiewicz University, Poznań, Poland
e-mail: natm <[email protected]>
The amylome is the name given to a set of proteins that are
capable of forming amyloid-like fibrils. A major factor is
the presence in the protein a segment that can form complementary beta sheets. [1]. The sequence and structure of
a model steric zipper was determined Eisenberg’s group. It
is hexapeptide with amino acid sequence NNQQNY and
was found in yeast prion protein Sup35 [2]. Similar structures are present in many proteins from all kingdoms of
life, but only some of them fulfill the conditions for effective steric zipper described above thanks to what not all of
these proteins exhibit amyloidogenic properties at physiological or only slightly disturbed conditions. The propensity of a given fragment of a protein to act a potential steric
zipper can be predicted by theoretical calculations using algorithm called Rosetta. The established energy threshold
for steric zipper peptides was set at –23kcal/mol [3].
Human cystatin C is a low molecular mass protein. It is
composed of 120 amino acid residues. It has 5 anti-parallel
β-stands and one α-helix. At physiological conditions is a
monomeric protein but under crystallization conditions
forms dimmer [4]. The first three-dimensional structure of
monomeric form of cystatin was determined for chicken
cystatin by X-ray crystallography and nuclear magnetic resonance spectroscopy (NMR). The conformation of hCC
was proposed to be very similar to chicken cystatin C what
was confirmed very recently of two of its mutants: V57N
[5] and stab1-hCC with new disulfide bonds ((L47C)–
(G69C)) [6]. The mechanizsm of the dimerization of hCC
was studied in depth and was shown to based on the three
dimensional domain swapping mechanism [7]. The 3D
profile method and calculated Rosetta algorithm values for
hCC show that in this protein there are two fragments, for
which high fibrillization propensity can be expected: fragment of loop L1 (Ala52-Asp65) and C-terminal part of the
molecule between Arg93 and Cys117. In this work results
of preliminary studies of nine peptides from the region
of loop L1, fulfilling potential conditions of steric zipper, will be presented.
References
1. Goldschmidt L et al. (2010) Proc Natl Acad USA 107: 3487-3492.
2. Sawaya MR et al. (2007) Nature 447: 453-457.
3. Nelson R et al. (2005) Nature 435: 773-778.
4. Grubb A (2000) Adv Clin Chem 35: 63-69.
5. Orlikowska M et al. (2011) J Struct Biol 173: 406-13.
6. Grzonka Z et al. (2001) Acta Biol Pol 48: 1-20.
7. Janowski R et al. (2001) Nat Struct Biol 8: 316-320.
Acknowledgements
Project co-funded by the European Union within the European Regional
Development Fund.
Reference
Rhee WJ et al. (2007) Biotechnol Prog 23: 1441-1446.
2nd Congress of Biochemistry and Cell Biology, September 5th–9th, 2011, Kraków, Poland
160
B8. Applications of X-ray crystallography in life sciences
P14.3
P14.4
Crystal structures of bovine and equine
serum albumins in complexes with drugs
Biochemical and structural characterization
of staphylococcal SplD protease
Anna Bujacz, Grzegorz Bujacz, B. Sekula
Przemyslaw Cichon1, Michal Zdzalik1, Magdalena Kalinska1,
Justyna-Stec Niemczyk2, Abber Jabaiah3, Benedykt
Wladyka2, Patric S. Daugherty3, Henning R. Stennicke4,
Adam Dubin2, Jan Potempa1, Grzegorz Dubin1
Technical University of Lodz, Institute of Technical Biochemistry, Lodz,
Poland
e-mail: Anna Bujacz <[email protected]>
Serum albumin is the main carrier for various substances
in the blood. It collects fatty acids from the digestive system, drugs from their place of application and hormones
from the secretion sites and delivers them to other parts of
the body. BSA and ESA, due to their wide accessibility, are
used as the human serum albumin (HSA) replacements in
many experiments, but they have only 75% and 76 % of
identity in respect to HSA. Serum albumin is a very universal carrier and to perform its task evolution equipped
it with a number of specialized binding sites. A single albumin molecule possesses 7 main fatty acid binding sites
and 4 additional ones with affinity only to short fatty acids.
There are 2 main drug binding sites that were identified,
and a few additional ones specific only to particular ligands,
among them: bilirubin, heme and steroid hormones. The
crystallographic studies of complexes of serum albumin
with ligands have so far been limited only to HSA. During
last 10 years more than 40 structures of HSA with ligands
were determined.
Many laboratories over the world attempted crystallization
and determining the structure of BSA. Finally, we have successfully established the crystallization conditions, solved
the apo BSA crystal structure, and started structural investigation of its complexes with ligands to compare them
with HSA analogs. Although ESA was one of the first proteins crystallized, its crystal structure was never deposited
in PDB. We found new crystallization conditions for ESA,
which are more suitable for creation of complexes and improve the resolution of diffraction data.
We present the crystal structures of bovine and equine serum albumins in complexes with drugs, which allows to
analyze the way they interact with this protein. Comparison
of the structures of the investigated serum albumins can
highlight the differences resulting from different sequence
and spatial specificity.
BSA is utilized in kinetic and affinity drug tests as a replacement of HSA as well as in many biochemical and pharmacological applications. One of the factors responsible for
the efficiency of drug transportation is the ability of the
drug to form a complex with serum albumin.
1Department of Microbiology and 2Department of Analytical
Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology,
Jagiellonian University, Krakow, Poland; 3Department of Chemical
Engineering, University of California at Santa Barbara, Santa Barbara, CA,
USA; 4Protein Engineering, Novo Nordisk A/S, Maaloev, Denmark
e-mail: Przemyslaw Cichon <[email protected]>
It was recently demonstrated that selected strains of staphylococci secrete up to six homologous serine proteases
grouped in a single spl operon. SplA, B and C proteases
were quite extensively characterized, but the properties of
SplD, E and F proteins remain unknown. Here we characterize the properties of one of those enzymes, the SplD
protease. The active protease was produced in recombinant
system in E. coli and purified to homogeneity. P1 substrate
specificity of the protease was determined using a combinatorial library of synthetic peptide substrates demonstrating exclusive preference for Thr, Ser, Leu, Ile, Ala and
Val. Following, a high-throughput cell surface protein substrate display and selection method (CLIPS) was utilized to
profile further subsites recognized by SplD. The analysis
demonstrated an unexpectedly narrow specificity of the
protease recognizing three consecutive residues with a consensus motif of (Y/W)(L/P)(L/T)↓S. In order to explain
the molecular basis of the restricted substrate specificity
we have crystallized the enzyme in two different conditions
and refined the structures to 1.6 Å and 2.1 Å resolution.
Analysis of obtained structures explains at atomic level the
P1 residue preference observed in biochemical assays but
does not clearly account for P2 or P3 specificity.
Acknowledgements
The project is supported by grant No. NN405 363939 from the Polish
Ministry of Science and Higher Education.
References
1. Sugio S et al. (1999) Protein Eng 2: 439-446.
2. Varshney A et al. (2010) Chirality 22: 77-87.
3. Ho JX et al. (1993) Eur J Bioch 215: 205-212.
2nd Congress of Biochemistry and Cell Biology, September 5th–9th, 2011, Kraków, Poland