The Skaggs Institute
for Chemical Biology
Structural Biology of Pheromones and Inteins by Nuclear Magnetic Resonance Spectroscopy
K. Wüthrich, M. Almeida, T. Etezady, M. Geralt, M.A. Johnson, W. Peti, W.J. Placzek
We use nuclear magnetic resonance (NMR) spectroscopy for structure determination and studies of correlations
between structure and function of proteins. The following sections illustrate our research activities
with 2 ongoing projects.
Pheromone Recognition in Euplotes Nobilii
Ciliated protozoa use combinations of 2 or several secreted, structurally closely related signaling proteins (pheromones)
to control vegetative growth and formation of mating pairs. The pheromone structures must contain
regions for recognizing mating-type signal and selectively signaling mitotic growth or the formation
of mating pairs, as well as regions for recognizing species. We determined the NMR solution structures
of 2 pheromones, En-1 and En-2, which had been purified from the Antarctic ciliated
protozoan Euplotes nobilii. The NMR structures revealed the locations of 4 disulfide
bonds in each protein, which previously could not be identified by biochemical methods. Comparison
with other pheromone structures provides insight into how the Euplotes pheromones are
used to identify mating types and differentiate between species. This collaboration with P. Luporini,
University of Camerino, Camerino, Italy, is currently being extended to include additional Euplotes
mating types. The long-term goal is to establish links between the mechanisms of signal transfer
among these single-cell, eukaryotic organisms and intercellular signaling of higher organisms
in health and disease.
Intein Structure and Function
Inteins are self-splicing protein elements that are encoded as insertions into various genes. When expressed in cells, inteins can excise
themselves from the surrounding protein in a remarkable multistep process that involves cleavage
of 2 peptide bonds to release the intein from the flanking regions and formation of a new peptide
bond that ligates the flanking regions to yield the mature, processed protein. Inteins have found
use in the generation of segmentally isotope-labeled proteins for NMR studies. This work thus
ties in closely with the many biomacromolecular NMR projects pursued within the Skaggs Institute.
The multistep mechanism of intein function starts with an acyl shift, which transfers the N-extein to the side chain of residue 1 of the intein
(serine or cysteine), concomitant with cleavage of the peptide bond at the intein N terminus (Fig.
|Fig. 1. Reaction scheme for the standard protein-splicing mechanism (depicted with serine at both splice junctions). Reproduced with permission from InBase, the Intein Database and Registry (http://www.neb.com/neb/inteins.html).
The second step is a transesterification in which the N-extein undergoes nucleophilic attack
by the side chain of the residue C-terminal to the intein (also serine or cysteine), yielding a protein
with two N termini known as the branched intermediate. With the N-terminal cleavage
thus completed, cleavage of the C-terminal peptide bond occurs by cyclization of the asparagine
residue at the C terminus of the intein. The resulting ester spontaneously undergoes an acyl shift
to form the more stable peptide bond and to yield the ligated mature protein as well as the excised
A subclass of inteins with alanine at position 1, rather than serine or cysteine, functions in a manner similar to that of standard inteins, although
alanine cannot act as a nucleophile for the initial acyl shift. To investigate the structural factors
that enable this alternative mechanism, we used multidimensional heteronuclear NMR spectroscopy
to determine the structure of the Methanococcus jannaschii KlbA intein. The protein adopts
a horseshoe-shaped fold consisting almost entirely of regular β-sheet
se condary structure (Fig. 2).
|Fig. 2. Ribbon representation of the NMR structure of the M jannaschii KlbA intein, with the extein residues glycine, G(1), and serine, S(+1), and the conserved block B histidine 96, H96, shown as stick drawings.
The N and C termini are found on extended strands that pass through
the center of the horseshoe, contributing to the formation of the active site along with residues
from other β-strands.
Several residues of a conserved sequence motif (block B) are involved in potentially activating
interactions with the scissile peptide bond. The amide group of alanine at position 1 forms a hydrogen
bond to the side-chain hydroxyl group of threonine at position 93, and the highly conserved histidine
at position 96, known to be involved in catalysis, is also within hydrogen-bonding distance (Fig.
|Fig. 3. Active-site residues in the M jannaschii KlbA intein. Hydrogen bonds are shown as dotted lines.
In the structure shown in Figures 2 and 3, the side chain of the C-extein nucleophile, serine (+1), which replaces the wild-type cysteine
residue, is located 10 Å from the scissile peptide bond at the N terminus, suggesting that
the intein would have to undergo a conformational change in order to catalyze cleavage of the N-terminal
peptide bond. Indeed, our studies revealed a slow conformational transition of the M jannaschii
KlbA intein in solution, which is completed within about 2 weeks at 55°C. The next steps in
this research, which is a collaboration with F.B. Perler, New England Biolabs, Ipswich, Massachusetts,
are to refine the structure of the second form of the intein and to investigate the mechanism of the
observed slow structural transition and its significance for the physiologic function of inteins.