Top view of porcine ribonuclease inhibitor, showing its horseshoe shape.[1] The outer layer is composed of
α-helices and the inner layer of
parallel β-strands. The inner and outer diameters are roughly 2.1 nm and 6.7 nm, respectively.
Ribonuclease inhibitor (RI) is a large (~450 residues, ~49 kDa), acidic (pI ~4.7),
leucine-rich repeatprotein that forms extremely tight complexes with certain
ribonucleases. It is a major cellular protein, comprising ~0.1% of all cellular protein by weight, and appears to play an important role in regulating the lifetime of
RNA.[2]
RI has a surprisingly high
cysteine content (~6.5%, cf. 1.7% in typical proteins) and is sensitive to oxidation. RI is also rich in
leucine (21.5%, compared to 9% in typical proteins) and commensurately lower in other hydrophobic residues, esp.
valine,
isoleucine,
methionine,
tyrosine, and
phenylalanine.
Structure
RI is the classic leucine-rich repeat protein, consisting of alternating
α-helices and
β-strands along its backbone. These
secondary structure elements wrap around in a curved, right-handed solenoid that resembles a
horseshoe. The parallel β-strands and α-helices form the inner and outer wall of the horseshoe, respectively. The structure appears to be stabilized by buried
asparagines at the base of each turn, as it passes from α-helix to β-strand. The αβ repeats alternate between 28 and 29 residues in length, effectively forming a 57-residue unit that corresponds to its genetic structure (each
exon codes for a 57-residue unit).
Binding to ribonucleases
The
affinity of RI for ribonucleases is among the highest for any
protein-protein interaction; the
dissociation constant of the RI-
RNase A complex is in the
femtomolar (fM) range under physiological conditions. Despite this high affinity, RI is able to bind a wide variety of RNases A despite their relatively low
sequence identity. Both biochemical studies and
crystallographic structures of RI-RNase A complexes suggest that the interaction is governed largely by
electrostatic interactions, but also involves substantial buried
surface area.[3][4] RI's affinity for ribonucleases is important, since many ribonucleases have
cytotoxic and
cytostatic effects that correlate well with ability to bind RI.[5]
Mammalian RIs are unable to bind certain pancreatic ribonuclease family members from other species. In particular,
amphibian RNases, such
ranpirnase and
amphinase from the
Northern leopard frog, escape mammalian RI and have been noted to have differential cytotoxicity against
cancer cells.[6]
Kobe B, Deisenhofer J (Mar 1995). "A structural basis of the interactions between leucine-rich repeats and protein ligands". Nature. 374 (6518): 183–6.
doi:
10.1038/374183a0.
PMID7877692.
S2CID4364436.
Kobe B, Deisenhofer J (Dec 1996). "Mechanism of ribonuclease inhibition by ribonuclease inhibitor protein based on the crystal structure of its complex with ribonuclease A". Journal of Molecular Biology. 264 (5): 1028–43.
doi:
10.1006/jmbi.1996.0694.
PMID9000628.
Suzuki M, Saxena SK, Boix E, Prill RJ, Vasandani VM, Ladner JE, Sung C, Youle RJ (Mar 1999). "Engineering receptor-mediated cytotoxicity into human ribonucleases by steric blockade of inhibitor interaction". Nature Biotechnology. 17 (3): 265–70.
doi:
10.1038/7010.
PMID10096294.
S2CID23140257.
Shapiro R, Ruiz-Gutierrez M, Chen CZ (Sep 2000). "Analysis of the interactions of human ribonuclease inhibitor with angiogenin and ribonuclease A by mutagenesis: importance of inhibitor residues inside versus outside the C-terminal "hot spot"". Journal of Molecular Biology. 302 (2): 497–519.
doi:
10.1006/jmbi.2000.4075.
PMID10970748.