1. Various Strategies Used to Obtain Proteins for
Crystallization and Biostructural Studies
Dorothee Ambrosius, R. Engh, F. Hesse,
M. Lanzendörfer, S. Palme, P. Rüger
Roche Pharmaceutical Research, Penzberg

2. Protein Classes extracellular proteins  plasma protein concentration:
 70 mg/ml
transporter (albumin)
immuno-globulin
enzymes, enzyme-inhibitors
coagulation factors, lipoproteins
 protein characteristics/
stability
often monomeric proteins
contain disulfide bridges
protease resistant
stable fold
intracellular proteins
 cytoplasma and organelles:
 mg/ml
multi-enzyme complexes
enzyme cascades
transcription complexes
focal adhesion/integrins
cytoskeleton, heat-shock proteins
 protein characteristics/stability
often multimeric complexes
no disulfide bridges
very labile proteins; short half-life
require stabilization: interaction with
other proteins

3. Protein Sources/Expression Systems

4. Biological Function of Cytokines
G-CSF
Neutrophils
Source: Herrmann/Lederle

5. Development Goals for Recombinant Human G-CSF
 Hu-G-CSF: hematopoietic growth factor (174 aa)
2 S-S bridges, one single Cys 17
 Clinical use: patients with neutropenia: after chemotherapy  improved haemotopoietic recovery
 reduction of infectious risks
 native sequence:  without additional N-terminal Met
 reduction of immunogenicity risk
 potency:  equal to Amgen´s Neupogen
 low production cost:  E. coli as host strain
 in vitro refolding
 consistent quality:  robust downstream scheme
 analytical methods established

6.  Strategy: Development of Recombinant Human G-CSF
Fusion Peptide
Human G-CSF
Fusion Peptide
high level expression
improved refolding
efficient separation of cleaved and uncleaved protein
optimized cleavage site
Protease
specific
efficient
recombinant
consistent quality
rhG-CSF
low production costs
without N-terminal Met
equal potency/efficiency
consistent quality
improved quality
Genetic engineering of an economic downstream process

7. Optimization of rhG-CSF Fusion Proteins
Fusion Peptide
Met G-CSF
Met-Thr-Pro-Leu  G-CSF
Met-Thr-Pro-Leu-His-His  G-CSF
Met-Thr-Pro-Leu-Lys-Lys  G-CSF
Met-Thr-Pro-Leu-Glu-Glu-Gly  G-CSF
Met-Thr-Pro-Leu-Glu-Glu-Gly-Thr-Pro-Leu  G-CSF
Met-Lys-Ala-Lys-Arg-Phe-Lys-Lys-His  G-CSF
Expression
(%)
100 30 25 10
Cleavage - ++ +
+++
Renaturation
(%) 10 20 50 90 80
Cleavage Site (Pro-Arg-Pro-Pro)
Source: EP ; DE

8. Refolding Kinetics of rhG-CSF Fusion Protein
Solubilization
6,0 M Gdn/HCl, pH 8.0
100 mM Tris,/HCl
100 mM DTE
1 mM EDTA
Temperature: RT
c= 20 mg/ml
Renaturation
0,8 M Arginine/HCl
100 mM Tris/HCl, pH 8.0
0.5 / 0.5 mM = GSH / GSSG
10 mM EDTA
Protein conc mg /ml
Time: 1- 2 hours
native
Pellet SN
denat.
Source: EP ; DE

9. Role of p53 in cell cycle control:“guardian of the genome”
cell cycle arrest:
repair defective genes
latent p53
active p53
cell type
level of p53
extent of DNA damage
genetic background
activation
accumulation
stress factors
or oncogenic proteins
mdm2
apoptosis:
kill harmful
deregulated cells
negative feedback loop !!

10. Engineering of MDM2 for biostructural purposes
The MDM2 oncoprotein is a cellular inhibitor of the p53 tumor
suppressor.
Goal: Improvement of biophysical properties of HDM2
(human MDM2) by “crystal engineering”
Known:  XDM2 (Xenopus laevis MDM2):
- better solubility, suitable for biostructural investigations
- wrong species and reduced binding affinity
 HDM2 (25-108):
- high binding affinity to p53 peptide
- prone to aggregation, not suitable for biostructural studies
Strategy:  use XDM2 as scaffold and humanize its p53-binding site
 introduce point mutations in HDM2 to increase solubility
 remove flexible ends at both sides of structured p53-binding
region

11. Structure of MDM2/p53-peptide complex
Figures taken from Kussie et al., Science 274 (1996) 948.
Resolution X-ray structures:
human MDM2/p53: 2.6 Å
Xenopus MDM2/p53: 2.3 Å
p53
mdm2
17-29
26-108

12. MDM2 variants created by protein engineering
human MDM2
p53 binding
HDM2 (17-125) X-ray published
HDM2 (25-108) X-ray
HDM2 (25-108) mutants X-ray
XDM2 (13-119) X-ray published, NMR
XDM2 (13-119) LHI NMR, X-ray
XDM2 (21-105) LHI X-ray
I50L
P92H
L95I

13. Human MDM2: Yields & Upscale
Step 15N-labeled non-labeled (LB)
(minimal medium)
Fermentation 10 L L
E. coli (wet weight) 90 g g
Inclusion bodies (w.w.) g g
IB total protein content g g
MDM2 (50-70% yield) g g
Renaturation (~25%) g g
MDM2 (Purification) g g
Final product g g

14. Crystals of hXDM/peptide
Some crystals comply with corporate identity rules
Patience might be rewarded
hXDM2/phage-peptide
hXDM2/p53 peptide
Conditions: 0.1 M MES pH 6.2, 4.0 M NaOOCH
3 days after micro seeding at 13 °C months at 4 °C

15. Protein Kinase Families (incomplete list)
I: Ser/Thr-Kinase Families Subfamilies/Structures
Ia: Non Receptor Ser/Thr-Kinase familiy
cAPK: cAMP dependent protein kinase PKA, PKB, PKC
cdks: Cyclin dependent kinase cdk2, cdk4, cdk6
MAPK: Mitogen activated protein kinase Erk, Erk2, Jnk, p38(,,)
MLCK: Myosine light chain kinase Twitchin, Titin
CK: Casein kinase Ck-1, Ck-2
PhK: Phosphorylase kinase (tetramer: , , , ) PhK
CaMK: Calcium/calmodulin dependen kinase CaMK
Ib: Receptor Ser/Thr-Kinase family
TGF1-R Kinase TGF1-ßR
II: Tyr-Kinase Families Subfamilies/Structures
IIa: Non receptor Tyr-Kinase family
SRC-family SRC, c-SRC, CSK, HCK
LCK: humam lymphocyte kinase: LCK, c-Abl
IIb: Receptor Tyr-Kinase family
EGFR-family: EGFR, ErbB2-4
InsR-family IRK, IGF1R, IRR
PDGFR-, CSFR-, Met-, Ron-familiy, FGF1-R, VEGFR-K
EphA1….EphB1, Trk A, B, C, etc.

16. PKA: 2 Å X-ray Structure
Further details for crystallization see poster of Ch. Breitenlechner
Figure 6: The X-ray structure of Protein Kinase A, shows the structural elements prototypical for the protein kinase family. An N-terminal beta strand (blue arrows) rich domain and a C-terminal alpha-helix (yellow cylinders) rich domain form the overall topology. The relative domain orientations are stablized by three interconnecting protein segments, ATP (here substituted by inhibitor AMP-PNP), and subtrate (here substituted by inhibitory peptide PKI). An activating phosphorylation site on the flexible activation loop causes structural rearrangements at the enzymatic activation site. The features generally present among protein kinases include flexible loop rearrangements controlled by phosphorylation, domain motions controlled by substrates or co-factors, and ATP binding at the domain interface. These features lead to heterogeneities of structure and phosphorylation, which generally hinder protein preparation for crystallization. On the other hand, they also contribute to the uniqueness of individual protein kinases and thus to the likelihood of finding drugs with the desired properties.

17. PKA: cyclic AMP Dependent Protein Kinase
Expression:  E. coli, solubly expressed in phosphorylated, active form
 mg purified protein (10 l fermentation)
Purification:  affinity chromatography with inhibitory peptide (PKI)
mimicking substrate binding
 Ref.: R. Engh & D. Bossemeyer, Adv. Enz. Reg. 41, 2001
Binding Affinity:  20 nM of inhibitory peptide (PKI)
Protein:  MW: 35 kDa
 Ser/The kinase
 monomeric 2 domain (C- and N-lobe) protein without
additional regulatory domains (SH2, SH3, etc.)
 extended structured C- and N-Terminus, which possibly
stabilizes the overall kinase structure
Ideal model: Ser/Thr protein kinase inhibitor studies
generation of other Ser/The kinase (e.g. PKB, Aurora) structures

18. Major Components of the Cell Cycle MachineryG0
mitogen induced progression
through the cell cycle requires
timely controlled activation of
different cyclin-dependent
kinases (CDKs)
cyclins (D, E, A, B), periodically
expressed throughout the
cycle, are the regulatory
subunits of CDKs (activation)
members of the p16(INK4)- and
p21(KIP)-protein family inhibit
CDKs and CDK-cyclin complexes
and arrest inappropriate cell
cycle progression
CDC2
INK4 M
cyclin B
Mitosis
CDK4/6
Cell Cycle
cyclin D G2 G1
CDC2
Kip/
Cip
DNA Replication
cyc. A/B
CDK2 S
cyclin E
CDK2
cyclin A
Kip/
Cip

19. Cyclin Dependent Kinases: CDK2 and CDK4/6
N. Pavletich, JMB 287, , 1999

20. Structural investigations of cdks (incomplete list)

21. Summary  Proteins show a tremendous diversity with respect to
- biological function and cellular location
- structure, conformation and stability
 E. coli is a very attractive expression system with respect to time, yield, costs
and production of isotope labeled proteins
 Application of in vitro protein refolding is a powerful tool to generate native
structured proteins and should be considered as alternative
 The protein kinase family is regulated by multiple mechanism and show
conformational diversity of catalytic cores; high degree of flexibility
- e.g. IRK(3P) and LCK (Tyr kinases) show structural homology to
cAPK and cdks (Ser/Thr kinases)
 Until today, most kinases successfully applied for structural research are
expressed as active P--enzyme in baculo/insect cells; exception PKA

22. Acknowledgement PEX: S. Kanzler, H. Brandstetter (MPI)
MDM2: G. Saalfrank, Ch. Breitenlechner (MPI), U. Jacob (MPI)
IL-16: B. Essig , P. Mühlhahn (MPI), T. Holak (MPI)
MIA: G. Saalfrank, C. Hergersberg, R. Stoll (MPI),
T. Holak (MPI)
cAPK: G. Achhammer, E. Liebig, Ch. Breitenlechner (MPI)
cdks: H. Hertenberger, J. Kluge, U. Jucknischke
G-CSF: S. Stammler, M. Leidenberger, U. Michaelis,
T. Zink (MPI), T. Holak (MPI)