Research Report 2000 - MDC

Structural Studies of
Proteins and Nucleic
Acids by X-ray
Crystallography
Udo Heinemann
We rely on macromolecular
crystallography to study structural
aspects of proteins and nucleic acids.
The crystal structures of these
molecules serve to explain their
function in biological processes,
conformational stability and folding.
General areas of interest include
nucleic acids and nucleic-acid binding
proteins, electron transport in
cytochrome P450 systems and the
structural determinants of the stability
and folding of globular proteins. Y.A.
Muller is engaged in studies of
hormone transport by the human sex
hormone-binding globulin and of
tissue factor. Many of these projects
involve collaborations with scientists
from Berlin and elsewhere. In
addition, there is a growing number of
in-house collaborations focussing, for
example, on Wnt signal transduction
involving β-catenin and conductin,
inhibitors of the transcription factor
NF-κB and G-protein coupled
receptors. In the newly developing
field of structural genomics, we have
helped create a Berlin-based research
project, the Protein Structure Factory
(PSF). Here, the aim is to set up a
local infrastructure for the semiautomated,
low-cost, high throughput
structure analysis of proteins. The PSF
contributes to a world-wide effort to
determine the structures of a
representative set of protein domains
that will greatly facilitate future
protein modelling and drug design
studies.
50
Nucleic acids and interacting
proteins
H. Delbrück, A. Diehl, O. Gaiser,
H. Lauble, U. Müller, Y. Roske,
E. Werner
The sequence-specific recognition of
nucleic-acid molecules by proteins
and other ligands is thought to be
mediated by local structural features
of the nucleic acid. We have
determined the crystal structures of
several synthetic RNA molecules in
an effort to identify the determinants
of specific protein binding. A chimeric
DNA-RNA hybrid, that corresponds
to the RNA-DNA junction formed
during minus-strand synthesis in the
course of reverse transcription of the
HIV-1 genome and carries specific
cleavage sites of the reverse
transcriptase-associated ribonuclease
H, has been shown to adopt the
standard A-type conformation. The
cleavage specificity of the
ribonuclease H has been suggested to
be associated with a structural
perturbation of the sugar-phosphate
backbone at the main cleavage site. In
another study, the crystal structure of
the acceptor stem helix of tRNA Ala
was determined at atomic resolution
from pseudo-merohedrally twinned
crystals. Here we have been able to
show that the G·U wobble base pair
known to be crucial for tRNA
recognition by the cognate tRNA
synthetase is hydrated in a
characteristic way and embedded in
the unperturbed, standard A-form
RNA. Significant progress has been
made in the structure analysis of
several nucleic-acid binding proteins.
The C-terminal domain of the
transcription factor KorB was
determined at high resolution and
shown to adopt a SH3-like fold
responsible for KorB dimer formation.
For the complex formed between the
C-terminal domain of translation
initiation factor IF2 and initiator
tRNA, crystallization and X-ray
diffraction conditions will have been
optimized to allow completion of the
structure analysis in the near future.
Electron transport in
cytochrome P450 systems
J.J. Müller
In vertebrates, enzymes of the
cytochrome P450 family catalyse a
variety of chemical reactions,
including steroid hormone
biosynthesis. They receive electrons
from a [2Fe-2S] ferredoxin which, in
turn, accepts electrons from an
NADPH reductase. We have
determined the crystal structure of
adrenodoxin, the ferredoxin from the
bovine adrenal gland mitochondrial
matrix, at 1.85 Å resolution (Figure
21). In spite of the low-level sequence
similarity, adrenodoxin bears close
structural similarity to the well known
class of plant-type [2Fe-2S]
ferredoxins and appears to share with
these proteins a common mode of
docking to the cognate reductase and
predicted electron transfer pathway.
Very recently, we have been able to
solve the crystal structure of the
chemically cross-linked complex of
adrenodoxin with adrenodoxin
reductase which will allow us to
model electron transfer between these
proteins with some confidence. The
crystal structures of adrenodoxin and
its complex with the adrenodoxin
reductase serve to explain a large
body of biochemical and mutational
data.
Structural basis of protein
stability and folding
J. Aÿ, A.M. Babu, H. Delbrück,
U. Müller
Selected aspects of protein folding
and thermodynamic stability can be
related to the native three-dimensional
protein structure as determined by Xray
crystallography. Over the last two
years, we have studied three different
model protein families in this respect.
Biochemical and crystallographic
analyses of 1,3-1,4-β-glucanases have
shown that the jellyroll fold of these
proteins resists various circular
permutations of the protein sequence
and, in the case of the engineered
protein GluXyn-1, even transplantation
of the autonomous folding unit of a
xylanase into a surface loop of the
protein. These studies have been
expanded using the protein
thiol/disulfide oxidoreductase DsbA,
where we have demonstrated by
crystal structure analysis that moving
the polypeptide chain termini from the
thioredoxin-like domain into the αhelical
domain by circular
permutation of the sequence has little
effect on the three-dimensional
protein structure. Finally, we are
currently investigating pairs of
bacterial cold-shock proteins of
closely similar sequence but
drastically different conformational