II. General Structure
III. Electrostatic Forces in
hRI:RNase 1 Recognition
IV. Key Complex-stabilizing
Residues of RNase 1
Applications of RI Evasion
(RNase) family is comprised of a
ubiquitous group of enzymes that share the
common ability to catalyze the cleavage of RNA. RNases play key roles
in the maturation
of many RNA molecules, including messenger RNAs and non-coding RNAs.
by RNase serves as a defense mechanism against RNA
viruses, provides the machinery for RNAi through the activity of the
Dicer and Drosha enzymes, and has been
recently targeted as a promising chemotherapeutic agent.
RNase activity can be detrimental when unregulated.
Intracellular RNAs that are not targeted for degradation are protected
from RNase activity by a number of different mechanisms. One mechanism
in particular is ribonuclease inhibitor (RI), which binds to
ribonucleases with the highest affinity of any protein-protein
interaction: the dissociation constant for the RI:RNase complex is ~20
under physiological conditions. In this tutorial, we report the
structure and chemical interactions within the human pancreatic
ribonuclease (RNase 1) and human ribonuclease inhibitor (hRI) complex.
ribonuclease inhibitor (hRI) is a 50 kDa cytosolic leucine-rich
repeat protein comprised
of 461 amino acids.
consists of a
remarkable pattern of alternating alpha
its backbone. These alternating secondary structures form a
solenoid, causing the overall structure of the protein to resemble a
inner wall of the
"horseshoe" is made entirely of beta
sheets, while the outer wall of the protein is comprised entirely of
hRI:RNase 1 complex forms an asymmetric unit through the
dimerization of two hRI molecules, each of which binds to one RNase 1 molecule
two hRI molecules
antiparallel beta sheet
through 24 residue-to-residue hydrogen bonds between the N-terminal
beta strand of each of the two molecules
1 is a 156 amino
acid protein that resembles the shape of a kidney.
active site of
RNase 1 is located in a cleft between the two main lobes of the
1 binds to hRI with
its active-site cleft covering the
C-terminal of hRI;
inserts into the cavity of the hRI
horseshoe, and the
other lobe lies over the face
of the horseshoe.
Because hRI is large and non-globular relative to RNase 1, it provides
an extensive binding area for the enzyme.
Electrostatic Forces in hRI:RNase 1 Recognition
total number of 23 residues of RNase 1 make contact with hRI.
13 of these residues
are responsible for the formation of 19 hydrogen bonds
that serve to stabilize the complex.
the active site of RNase 1 also make contact with hRI:
His119. Hydrogen bonding of
Lys41 with hRI is the strongest and most
conserved interaction between hRI and other RNase variants. This
interaction contributes most extensively to RNase 1 inhibition upon
binding to hRI.
regions of high shape complementarity
between residues of hRI and RNase 1 are ultimately responsible for the
protein-protein interaction affinity in the femtomolar range.
in the C-terminal of hRI and
tryptophans 261, 263 and 318
of the 11-12 o’clock region of the horseshoe contribute a
particularly high amount of shape complementarity. These regions allow
an extensive number of hydrogen bonds to be made between hRI and RNase
that serve to stabilize the complex and increase the efficiency of
Key Complex-stabilizing Residues of RNase 1
all of the residues responsibe for intermolecular hydrogen bonding
between regions of high shape complementarity in the hRI:RNase 1
complex, three residues in particular provide the highest energetic
contribution to complex stabilization.
residues include Arg39,Asn67
of RNase 1 makes
hydrogen bond with the side
Glu401 and a main-chain
with the main chain of
hRI. The energetic consequence of an R39D substitution is 2.1 kcal/mol,
making this contact the second most important contribution to complex
of RNase 1 forms a main chain
hydrogen bond to
Tyr437 and makes
van der Waals contact with
Gly410 of hRI. A less
contributor to complex stability, the consequence of an N67D mutation
is 1.9 kcal/mol.
has the greatest
influence on the stability of the hRI:RNase 1
complex, 2.7 kcal/mol.
Arg91 makes contact with the
surface of hRI, forming two
Chemotherapeutic Applications of RI Evasion
cytotoxicity has shown potential to serve as an effective
chemotherapeutic agent (Rutkoski & Raines, 2008). An amphibian
member of the RNase A family called Onconase (ONC) is currently in
phase III trials for the treatment of malignant mesothelioma. The human
RNase 1 has an increased therapeutic advantage over ONC through
greater catalytic activity and decreased immunogenicity. However, the
cytotoxicity of RNase 1 and other ribonuclease variants is impeded by
their strong affinity for RI, the basis of which we have described in
this tutorial. Engineering ribonucleases to have decreased affinity for
RI while leaving their catalytic activity undisturbed will be a
critical strategy for using ribonucleases as chemotherapeutic agents in
variant of RNase 1 has recently been engineered by Johnson et al (2007)
with a 5 x 109 -fold decrease in RI affinity, consequently making it
the most RI evasive ribonuclease to date.
R91D are responsible for this
in RI affinity. Three of the key wild-type residues responsible for the
extraordinary binding affinity of RNase 1 to hRI were reported in this
tutorial, Arg39, Asn67 and Arg91.
Johnson, McCoy G. Jason, Bingman A.
Craig, Phillips Jr N.
George, and Raines T. Ronald. "Inhibition of Human Pancreatic
Ribonuclease by the Human Ribonuclease Inhibitor Protein." Journal of
Molecular Biology 368 (2007): 434-49. Web.
Dickson A., Haigis C. Marcia, and Raines T. Ronald. "Ribonuclease
Inhibitor: Structure and Function." National Institutes of Health 80
(2010): 349-74. Web.
Anastassios C., Shapiro, Robert, Acharya, Ravi K. “Molecular
recognition of human angiogenin by placental ribonuclease
inhibitor—an X-ray crystallographic study at 2.0 Å
resolution.” The EMBO Journal: (1997) 16, 5162-
Rutkoski J., and Raines T. Ronald. "Evasion of Ribonuclease Inhibitor
as a Determinant of Ribonuclease Cytotoxicity." National Institutes of
Health 9.3 (2010): 185-99. Web.