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¡¡ Robert Shapiro ¡¡ Structure
and function of angiogenin Human
angiogenin (Ang) is an unusual member of the pancreatic RNase superfamily that
induces blood vessel formation in vivo. Although
Ang contains counterparts for the key catalytic residues of bovine pancreatic
RNase A, it cleaves standard RNase substrates 105 - 106
times less efficiently than does RNase A. Despite
this apparent weakness, the enzymatic activity of Ang appears to be essential
for biological activity: replacements
of important active site residues invariably diminish ribonucleolytic and
angiogenic activities in parallel, and a substitution that increases enzymatic
activity also enhances angiogenic potency.
Crystal structures of two of the inactive variants show that there are no
significant changes beyond the replacement site, indicating that loss of
biological activity is directly attributable to disruption of the catalytic
apparatus. The identities of the in
vivo substrates or ligands of Ang are now being investigated by the research
group of Dr. Guo-fu Hu at this Center. A
major focus of our studies has been to understand the structural basis for the
unique enzymatic properties of Ang. Our
approach combines site-directed mutagenesis, kinetic mapping, and X-ray
crystallography (in collaboration with Prof.
K. R. Acharya of the University of Bath, U.K.). Thus far, the roles
of many active site components in catalysis, binding, or modulation of activity
have been determined. In addition,
several surprising structural features of Ang that contribute to the attenuation
of enzymatic activity have been revealed. The
most striking of these is the obstruction of the putative pyrimidine-binding
site by Gln117, which lies on the C-terminal 310 helix.
Modeling demonstrates that a conformational change to open this site is
required in order for Ang to bind and cleave RNA substrates: i.e., that the
native Ang structure observed by crystallography and NMR is inactive.
This raises the possibility that Ang undergoes activation at the
appropriate time and location in vivo by binding to other cellular components or
through post-translational modification. We
are now working with crystallographer K. R. Acharya and NMR spectroscopist F. Ni
(National Research Council of Canada Biotechnology Research Institute) to define
the active conformation of Ang by determining structures of superactive Ang
variants and complexes of Ang with pyrimidine-containing inhibitors. Development
of small-molecule inhibitors of angiogenin
Angiogenin was first isolated from medium conditioned by human colon
adenocarcinoma cells, and has been shown by Drs. Karen Olson
and James Fett at this Center to play a critical role in the establishment
and/or metastatic spread of a wide range of human tumor xenografts in athymic
mice, most likely by contributing to tumor angiogenesis. Moreover, Ang expression has been found to be elevated in
numerous types of human cancers, and in many instances a specific association of
Ang with cancer aggressiveness and/or progression has been demonstrated.
These findings identify Ang as an attractive target for anticancer
therapy.
One strategy for the development of Ang antagonists is to design
molecules that bind tightly to the ribonucleolytic active center of the protein,
which has been demonstrated to be a key actor in the angiogenic mechanism.
Part of our effort in this area has focused on nucleotide-based
compounds, and has identified several Ang inhibitors that may be useful as
starting points for structure-based design.
Parallel work has been performed with RNase A as a model system that is
more amenable to crystallographic study at this stage.
Recently, we have been able to expand our approach to include
non-nucleotide compounds through a new high-throughput assay, which is being
used to screen large compound libraries in collaboration with the Harvard
Institute for Chemistry and Cell Biology. Several
new leads have been identified that bind much more tightly than any of the
nucleotides. These will now be used
for rational design of improvements based on models of ligand complexes and
subsequently actual 3D structures to be determined by our collaborators K. R.
Acharya and F. Ni. Predictions of
binding modes of inhibitors and the energetic effects of proposed modifications
are performed with the programs AutoDock and LUDI, respectively; AutoDock has
been validated using several difficult test cases from the literature where
minor alterations of inhibitor structures caused major unexpected changes in
binding modes. Human
placental RNase inhibitor
Human placental RNase inhibitor (RI) is a 50-kDa cytosolic protein that
binds mammalian pancreatic RNase superfamily enzymes with dissociation constants
(Kd values) ranging from 10-13 M to below 10-15
M. Its tightest-binding natural
ligand is angiogenin (Ang), with a Kd value of 0.5 fM
reflecting extremely slow dissociation of the complex (t½ = 73
days) and rapid association that approaches the diffusion limit. hRI is constructed almost entirely of tandem alternating 29-
and 28-residue leucine-rich repeat (LRR) units with average 40% sequence
identity. LRRs are common motifs
utilized for protein-protein interactions and have been found in more than 70
proteins with diverse functions, including signal transduction, cell cycle
control, cell adhesion, and enzyme regulation.
The interactions of RI with its various ligands provides a particularly
intriguing and powerful system for studying the structural basis for molecular
recognition in general and the function of LRRs in particular because of the
extraordinarily high affinities achieved and the broad specificity of RI for
proteins that typically share only 25-35% sequence identity.
Moreover, suitably engineered derivatives of RI (with greater selectivity
for Ang and increased extracellular stability) might be useful as therapeutic
agents for treatment of cancer and other angiogenin-dependent diseases.
We are investigating the molecular basis for tight-binding of RI to Ang,
RNase A, and other RNases by single-site and multi-site mutagenesis, together
with X-ray crystallography (in collaboration with Prof.
K. R. Acharya of the University of Bath, U.K.).
The crystal structures of the complexes of porcine RI with RNase A
(determined by Kobe and Deisenhofer) and human RI with Ang (determined by the
Acharya laboratory) show that both interfaces are large, and that the ligands
dock similarly, although few of the specific interactions formed are analogous. Mutational
analysis of the Ang complex, however, reveals that the binding energy is focused
largely in a single relatively small "hot spot" containing residues
from the C-terminal region of RI and the active site of the enzyme; only one
other part of the interface, a region rich in tryptophans, makes an important
energetic contribution. Within both
the hot spot and Trp-rich regions, residues function cooperatively such that
mutational effects are superadditive, even in many instances where there are no
obvious structural linkages. The
RI-RNase A complex contains a similarly-positioned hot spot, but only one major
contact (involving the catalytic lysine of the enzyme) appears to be conserved.
The binding energy is much more widely distributed over the interface in
this complex, and the combined effects of replacing multiple residues are
markedly subadditive, rather than superadditive.
Thus, RI recognizes Ang and RNase A in largely distinctive ways.
Efforts are now underway to determine whether the general
"themes" suggested by the RI-Ang and RI-RNase A complexes (e.g.,
anchoring of the ligand through contacts with the active site; the use of
cooperativity) apply to the interactions of the inhibitor with other ligands,
such as human RNase 2 (also known as eosinophil-derived neurotoxin).
Additional areas being actively pursued are the generation of
"minimized" RI derivatives based on the hot spot region and the
investigation of the role of the 32 cysteine residues of RI in conferring
sensitivity to oxidation. Current
research group Jeremy
L. Jenkins, Ph. D. --
Structure-based design of angiogenin inhibitors Kapil
Kumar, Ph. D. -- Engineering and minimization of protein RNase inhibitor Matthew
Crawford, B. S. -- Stabilization of protein RNase inhibitor Previous
group members (since 1996) Richard
Kao, Ph. D. Daniel
P. Teufel (visiting student from University of Bath) Melisa
Ruiz-Gutierrez, B. S. Anwar
Jardine, Ph. D. Marsha
Crochierre, B. S. Chang-Zheng
Chen, Ph. D. Cecilia
Roh, B. S. Recent
publications Russo
A, Acharya KR, Shapiro R. Small
molecule inhibitors of pancreatic and related RNases.
Methods in Enzymology. In
Press. Shapiro
R. Cytoplasmic RNase inhibitor.
Methods in Enzymology. In
Press. Holloway
DE, Hares MC, Shapiro R, Subramanian V, Acharya KR High level expression of three members of the murine
angiogenin family in Escherichia coli and purification of the recombinant
proteins. Protein Expression and
Purification 2001. In Press. Riordan
JF, Shapiro R. Isolation and
enzymatic activity of angiogenin. In:
Schein CH, ed. Methods in Molecular Biology, Vol. 160: Nuclease Methods and
Protocols. Humana Press,
2001: 375-385. Shapiro
R, Ruiz-Gutierrez M, Chen C-Z. Analysis
of the interactions of human ribonuclease inhibitor with angiogenin and
ribonuclease A by mutagenesis: Importance
of inhibitor residues inside vs. outside the C-terminal ¡°hot spot¡±.
J Mol Biol 2000; 302:497-519. Chen
C-Z, Shapiro R. Superadditive and
subadditive effects of ¡°hot spot¡± mutations within the interfaces of
placental ribonuclease inhibitor with angiogenin and ribonuclease A.
Biochemistry 1999;29:9273-9285. Leonidas
DD, Shapiro R, Irons LI, Russo R, Acharya KR.
Towards rational design of ribonuclease inhibitors:
High-resolution crystal structure of a ribonuclease A complex with a
potent 3N,5N-pyrophosphate-linked dinucleotide inhibitor.
Biochemistry 1999;32: 10287-10297. Leonidas
DD, Shapiro R, Allen SC, Subbarao GV, Veluraja K, Acharya KR.
Refined crystal structures of native human angiogenin and two active site
variants: Implications for the
unique functional properties of an enzyme involved in neovascularization during
tumour growth. J Mol Biol
1999;285:1209-1233. Russo
N, Shapiro R. Potent inhibition of
mammalian ribonucleases by 3N,5N-pyrophosphate-linked nucleotides.
J Biol Chem 1999;274:14902-14908. Fu
X, Roberts WG, Nobile V, Shapiro R, Kamps MP.
mAngiogenin-3, a target gene of oncoprotein E2a-Pbx1, encodes a
new angiogenic member of the angiogenin family. Growth Factors 1999;17:125-137. Yamaguchi
N, Anand-Apte B, Lee M, Sasaki T, Fukai N, Shapiro R, Que I, Lowik C, Timpl R,
Olsen BR. Endostatin inhibits VEGF-induced
endothelial cell migration and tumor growth independently of zinc binding.
EMBO J 1999;18:4414-4423. Shapiro
R. Structural features that
determine the enzymatic potency and specificity of human angiogenin:
Threonine-80 and residues 58-70 and 116-123.
Biochemistry 1998; 37:6847-6856. Acharya
KR, Leonidas DD, Papageorgiou AC, Russo N, Shapiro R. Structural studies on angiogenin, a protein implicated in
neovascularization during tumor growth. In:
Margoudakis M, ed. Angiogenesis: Models, Modulators, and Clinical
Applications. NATO ASI Series, Vol. 298, 1998:165-178. Ding
Y-H, Javaherian K, Lo K-M, Chopra R, Boehm T, Lanciotti J, Harris BA, Li Y,
Shapiro R, Hohenester E, Timpl R, Folkman J, Wiley DC.
Zinc-dependent dimers observed in crystals of human endostatin.
Proc Natl Acad Sci USA 1998;95:10443-10448. Boehm
T, O¡¯ Reilly MS, Keough K, Shiloach J, Shapiro R, Folkman J.
Zinc-binding of endostatin is essential for its antiangiogenic activity.
Biochem Biophys Res Commun 1998;252:190-194. Papageorgiou
AC, Shapiro R, Acharya KA. Molecular
recognition of human angiogenin by placental ribonuclease inhibitor - an X-ray
crystallographic study at 2.0 Å resolution.
EMBO J 1997;16:5162-5177. Russo
N, Shapiro R, Vallee BL. 5N-Diphosphoadenosine
3N-phosphate is a potent inhibitor of bovine pancreatic ribonuclease A.
Biochem Biophys Res Commun 1997;231:671-674. Chen
C-Z, Shapiro R. Site-specific
mutagenesis reveals differences in the structural bases for tight binding of
RNase inhibitor to angiogenin and RNase A.
Proc Natl Acad Sci USA 1997; 94:1761-1766. Leonidas
DD, Shapiro R, Irons LI, Russo N, Acharya KA.
Crystal structures of
ribonuclease A complexes with 5N-diphosphoadenosine 3N-phosphate and
5N-diphosphoadenosine 2N-phosphate at 1.7 Å resolution.
Biochemistry 1997;36:5578-5588. Soncin
F, Strydom DJ, Shapiro R. Interaction
of heparin with human angiogenin. J
Biol Chem 1997;272:9818-9824. Russo
N, Acharya KR, Vallee BL, Shapiro R. A
combined kinetic and modeling study of the catalytic center subsites of human
angiogenin. Proc Natl Acad Sci USA
1996;93:804-808. Nobile
V, Vallee BL, Shapiro R. Characterization
of mouse angiogenin-related protein: Implications
for functional studies on angiogenin. Proc
Natl Acad Sci USA 1996;93: 4331-4335 Shapiro
R, Riordan JF, Vallee BL. LRRning
the RIte of springs. Nature
Structural Biology 1995;2:350-354. Acharya
KR, Shapiro R, Riordan JF, Vallee BL. Crystal
structure of bovine angiogenin at 1.5-Å resolution.
Proc Natl Acad Sci USA 1995;92:2949-2953. Papageorgiou
AC, Acharya KR, Shapiro R, Passalacqua EF, Brehm RD, Tranter HS.
Crystal structure of the superantigen enterotoxin C2 from Staphylococcus
aureus reveals a zinc-binding site. Structure
1995;3:769-779. Brown
WE, Nobile V, Subramanian V, Shapiro R. Mouse
angiogenin gene family: Structures
of an angiogenin-related protein gene and two pseudogenes.
Genomics 1995;29:200-206. Soncin
F, Shapiro R, Fett JW. A cell-surface proteoglycan mediates human adenocarcinoma HT-29 cell adhesion to human angiogenin.
J Biol Chem 1994;269:
8999-9005. Acharya
KR, Shapiro R, Allen SC, Riordan JF, Vallee BL. Crystal structure of human angiogenin reveals the structural
basis for its functional divergence from ribonuclease.
Proc Natl Acad Sci USA 1994;91:2915-2919. Russo
N, Shapiro R, Acharya KR, Riordan JF, Vallee BL.
Role of glutamine-117 in the ribonucleolytic activity of human angiogenin.
Proc Natl Acad Sci USA 1994;91:2920-2924. |