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Structural Studies of Rio2, an Atypical Serine Kinase Required for Ribosome
Biogenesis
LaRonde-LeBlanc
N and Wlodawer A. Crystal structure of A. fulgidus Rio2 defines a new
family of serine protein kinases. Structure 12: 1585–94, 2004.
 ibosome
production is fundamental to cellular proliferation and therefore to tumorigenesis.
Increased nucleolar size, which corresponds to increased ribosomal RNA (rRNA)
production, has long been accepted as a hallmark of tumor cells. rRNA processing
is a stepwise process that requires several non-ribosomal factors. Yeast Rio1,
one such factor, is the founding member of the RIO kinase family. Yeast Rio1
is an essential gene for proper cell cycle progression and chromosome maintenance
in addition to rRNA processing (Angermayr M et al. Mol Microbiol 44:
309–24, 2002; Vanrobays E et al. Mol Cell Biol 23: 2083–95,
2003). Sequence alignments have demonstrated that members of two RIO subfamilies,
Rio1 and Rio2, are represented in organisms across the biological spectrum.
The two subfamilies are distinct in several highly conserved regions of the
catalytic domain, known as subdomains—which have been shown to be
important for the fold of the domain, or for ATP binding and phosphoryl transfer
catalysis. For example, Rio1 and Rio2 each contain a distinct nucleotide-binding
loop in what is known as subdomain I. In addition, the Rio2 subfamily is characterized
by a conserved N-terminal domain that is not present in Rio1.
The RIO kinases are interesting molecules for several reasons. They are ancient
essential proteins, and it has been suggested that they represent an evolutionary
link between prokaryotic lipid kinases and eukaryotic protein kinases (ePKs).
They are very divergent in sequence from known protein kinases and lack many
of the sequence features required for the function of ePKs. They are rRNA processing
factors with kinase activity and, thus, are attractive potential targets for
therapeutic intervention. To understand the nature of these enzymes and their
relationships to ePKs, we determined the X-ray crystal structure of Rio2 from
Archaeoglobus fulgidus, a hyperthermophilic archaeal organism. Despite
a lack of significant sequence homology to ePKs, the RIO catalytic domain bears
striking structural homology to the canonical kinase domain (Figure
1). The catalytic domain of kinases such as protein kinase A (PKA) contains
11 subdomains. The catalytic domains of members of the RIO kinase family contain
only eight, and these subdomains contain variations that produce differences
in phosphate binding, and perhaps substrate recognition and catalysis as well.
The most significant difference is the complete absence of subdomain VIII, which
is known as the APE (Ala-Pro-Glu), or activation loop. In ePKs, the APE loop
is often phosphorylated to regulate the activity of the kinase and provides
much of the surface for binding to substrate peptide. The lack of this region
in the catalytic domain of Rio2 and other RIO kinases leaves open the question
of how RIO kinases bind their substrates. In addition, we found that the N-terminal
Rio2–specific domain adopts a winged helix fold, which is commonly used
by proteins for nucleic acid interactions.
Figure 1. The structure of atypical serine kinase Rio2. A ribbon
illustration of the three-dimensional structure of the Rio2-ATP-Mn complex showing
the secondary structure elements and the various domains. DFG, Asp-Phe-Gly.
Our studies provided a detailed description of the ATP-binding pocket of the
Rio2 proteins. To determine the mode of ATP binding for the Rio2 kinase, we
soaked crystals of the Rio2 protein in a solution containing an ATP analog,
AMPPNP, and Mn2+
ions. Divalent cations are known to be required for catalytic activity, and
RIO kinases retain the metal-binding residues. Although we were able to observe
AMPPNP bound in the active site, we did not find any metal ions. We therefore
concluded that the conformation of the complex was not a productive one, and
that the confines of the crystal did not allow for the conformational changes
required for proper binding of ATP and metal ions. More recent studies confirmed
this conclusion. Crystals of the Rio2-ATP-Mn2+
complex assembled before crystallization contain the ATP molecule as well as
two Mn2+ ions in
the active site. We now know that ATP binds in the RIO kinase domains in a conformation
that is different from canonical ePKs. Due to this difference, the γ-phosphate
is located in a position that would require further conformational changes in
the Rio2 protein for substrate access. These findings support our conclusion
that the peptide substrate binding by RIO kinases must differ significantly
from that of canonical serine kinases.
Because the RIO kinases lack the classical peptide substrate–binding loops
and bind ATP in a distinct conformation, it is likely that their inhibitors
will be very distinct from the inhibitors of classical protein kinases. Our
investigations should be helpful in guiding the efforts to develop such inhibitors,
based on the structures of the enzymes from each RIO subfamily with bound nucleotides,
as well as on the planned structural studies of their complexes with peptides.
These discoveries have implications for the role of RIO proteins in rRNA cleavage
and provide a framework for experiments to determine their target and function.
In addition, this work defines, in structural terms, a novel family of atypical
protein serine kinases.
Nicole LaRonde-LeBlanc, PhD
Postdoctoral Fellow
Macromolecular Crystallography Laboratory
NCI-Frederick, Bldg. 539/Rm. 145
Tel: 301-846-5326
Fax: 301-846-5326
nicole@ncifcrf.gov
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