KH3 K-Homology RNA-Binding Domain
Kenny Farabaugh '10
Nova-1 is an autoantigen in the human autoimmune
neurological disease paraneoplastic opsoclonus-myclonus ataxia (POMA).
POMA is categorized by loss of motor control of limbs and
eyes. Ordinarily, the Nova-1 protein is only expressed in the
central nervous system (CNS), but in POMA patients, it is expressed in
tumors outside the CNS, causing an autoimmune response against the
Nova-1 protein, which is considered 'non-self' by virtue of being found
outside the CNS. The resulting autoimmune attack on the CNS
is believed to cause the loss of motor control characteristic of this
Nova-1 is involved in the regulation of alternative splicing of mRNA in
neural cells. This function was originally theorized based on
the proximity of Nova-1 binding sites to introns near known
alternatively spliced exons. The mechanism
selective binding of the single-stranded hairpin loop of an
RNA molecule, performed by the three K-Homology (KH) domains of the
Nova-1 protein. When Nova-1 is present, the KH domains
bind a specific glycine receptor protein pre-mRNA with a repeated UCAU
sequence tetrad, thereby inhibiting splicing at
this site and causing splicing at
another site. When the autoimmune response is activated,
antibodies are produced that preferentially bind the KH domain of
Nova-1, inhibiting RNA-binding; when this alternative splicing does not
the result is apoptotic cell death and the POMA disease. 
the entire Nova-1 protein has not been crystallized, but the most
important functional domains, the KH RNA-binding domains, have.
Since their discovery in the heterogeneous nuclear
ribonucleoprotein (hnRNP) K, a superfamily of homologous domains have
been found in eukaryotes and eubacteria, including such proteins
as insulin-like growth factor 2 mRNA-binding protein, Fragile X disease
protein FMR-1, and the 40S
ribosomal protein S3. The
functions of proteins containing KH
RNA-binding domains ranges from translation to alternative splicing to
mRNA localization and possibly even RNA interference. 
II. General Structure 
The KH domain contains 70 amino acid
residues on average across the superfamily; specifically, the Nova-1
KH3 domain is composed of 76
residues, with 3 antiparallel
beta-ribbons on one side
and 3 alpha-helices
other in the order S1-H1-H2-S2-S3-H3.
of KH domains includes two
well-defined loops, the H1-H2
loop and the S2-S3
loop . The H1-H2
a highly conserved Gly-X-X-Gly
section, where X is usually Arg, Lys, or Gly.
In the Nova-1 KH domains, X represents Lys and Gly (residues 23-24).
The S2-S3 loop
is incredibly variable among the KH
superfamily, and has been known to include up to 44 amino acid
residues. In the Nova-1 KH domain, the S2-S3 loop contains 11 amino
The KH domain is structured in such a way that
aliphatic hydrophobic amino acid
residues mainly face inward
residues face outward
results in a tightly structured domain
that resists both rearrangement and protease degradation in a
This is how the KH1
and KH2 domains are
believed to fit into the
entire Nova-1 protein.
5, 6] We
see that the H1-H2 loops
protrude from the outer face of the
molecule, allowing Nova-1 to bind the RNA with no steric interference
from the rest of
the protein. The KH3 domain most likely protrudes from the Nova-1 in
the same way.
III. RNA Binding 
The most important function of the Nova-1 KH
is binding the single-stranded hairpin loop of an mRNA, preferentially
those with a repeated UCAU tetrad. The crystal structure was
created using this base repeat, but we can theorize similar
interactions occuring between RNA bases
U-13, C-14, A-15, and
loop, sometimes referred to as the hydrophobic binding platform, Gly-22, Lys-23,
Gly-24, and Gly-25.
Electrostatic interactions include hydrogen-bonding in the H1-H2 loop ,
specifically between the nitrogen on the side chain of Lys 23 and an
oxygen on U-13
nitrogen on the backbone of Gly-22
and the oxygen of the pentose
sugar ring of U-13
the nitrogen on the backbone of Gly-24
an oxygen on the
phosphate backbone of C-14
the nitrogen on the backbone of of Gly-25
and the oxygen in the pentose sugar ring of A-15
These Gly residues are highly
conserved because of their size - larger residues would disrupt the
chain and eliminate the functioning domain of the protein. Lys-40
can form a hydrogen bond with an
oxygen on C-16, the
only interaction with this base at all,
the argument that the specificity is not as pronounced for this base as
for the preferred uracil
Ser-19 , Leu-41
, and Arg-54
form hydrogen bonds with an oxygen on
U-13 a nitrogen in
A-15, and a nitrogen or an oxygen in C-14, respectively.
Nonspecific hydrophobic Van der Waals
interactions can occur
between Ile-21 and the
aliphatic segment of Lys-43,
and the ring structures of C-14
are not as strong as the hydrogen bonds, but they may help account for
the specificity of the KH-domain binding the UCAU RNA-base tetrad.
KH domains - Nova-1
has three, and some include as many as fifteen! The KH
can intereact with each other not only to increase RNA-binding
specificity but also to bind more than one mRNA
The junction of the KH monomers occurs between the N-terminal S1 beta ribbons
each, lining them up to continue the antiparallel
The dimer is stabilized by hydrophobic
interactions (more Van
der Waals forces), including the Tyr-6,
Phe-7, Leu-8, Lys-9, Leu-11, Pro-13, Ala-103, Val-106, Ile-108,
Ile-109, Val-110, ,
Hydrophobic forces also hold the dimer together in the H3 helix,
evident in the Ile-62, Ala-73,
Ile-76, Gly-157, Pro-159, Val-166, Ile-169, Ile-173, and Pro-177
In the KH domain superfamily, a number
of hydrophobic amino acid residues are highly conserved,
such as Glu-6, Val-8, and
Unfortunately, these residues are not conserved in the Nova-1
KH domain. This general conservation in the superfamily
supports the formation of the dimer as well as showing that the dimer
is potentially biologically important in evolved species.
It has been theorized that the Nova-1 KH domain can even form a tetramer
This tetramer would be an asymmetric unit, and
therefore noncrystallographic, but the formation is feasible.
This feat would help explain why certain KH domain-containing
proteins have numerous copies.
The structure of
the Nova-1 KH RNA-binding domain provided some insights into other
RNA-binding proteins. Many RNA binding proteins
contain a similar alpha helix/beta sheet structure.
sheets have been found to be involved in binding more
single-stranded RNA-binding proteins, such as in the U1 snRNP A, the
MS2 phage protein coat, and several tRNA synthetases. The
alpha helices have been found to be involved in binding
more often in
double-stranded RNA-binding proteins, such as the HIV-1 Rev binding to
the major groove of RRE RNA.
KH domains in
other proteins in C.
elegans resulted in early identification of several
loss-of-function mutations. Mutation of the first Gly in the
Gly-X-X-Gly H1-H2 loop
results in loss-of-function because the KH
domain can no longer bind the RNA. Surprisingly, some
mutations in the variable region can lead to loss of function as well,
such as the KH domain of Drosophila
Bicaudal C protein; it is thought that this mutation disrupts protein
folding and binding with correlated KH domains.
Gly-X-X-Gly H1-H2 loop is
so well conserved
all KH domains, it
is considered the most important part of the structure.
However, most of the hydrogen bonds that form between the two
molecules involve the phosphate backbone or the pentose sugars, which
have no specificity factor. What then, accounts for the
Gly-X-X-Gly specifically targeting the UCAU RNA-base tetrad?
It has been theorized that because most proteins with a KH
domain contain more than one domain linked together by another loop,
the domains may work in tandem to bind specific RNA molecules with a
much longer UCAU repeat in its secondary structure. Future
studies may indicate the nature of specific RNA-binding by multiple
correlated KH domains.
Chen, Hua, Edo, Carme, Buckanovich, Ronald J., Yang, Yolanda YL,
Musunuru, Kiran, Zhong, Ru, Darnell, Robert B., and Burley, Stephen K.
February 1999. Crystal structures of Nova-1 and
Nova-2 K-homology RNA-binding domains. Structure.
release. Rockefeller University researchers identify
regulates RNA in nerve tissue. February 24, 2004.
The Rockefeller University. Copyright 2004-2005.
(Accessed 9 December, 2007)
Wilce, J. A., Vivian, J. P., Porter, C. J., Barker, A., Leedman, P. J.,
and Wilce, M. C. J. February 2005. Structure and
RNA binding of the third KH domain of poly(C)-binding protein 1.
Acids Research. 33(4):1213-1221.
Kopp J., Guex N., and
Peitsch MC (2003) SWISS-MODEL: an automated protein homology-modeling
Acids Research. 31:3381-3385.
Guex, N., and Peitsch, M.C. (1997) SWISS-MODEL and the
Swiss-PdbViewer: An evironment for comparative protein
Arnold, K.,Bordoli, L., Kopp, J., and Schwede, T.
(2006). The SWISS-MODEL Workspace: A
web-based environment for protein structure homology modelling.