Visualization of transient encounter complexes in
Chun Tang1, Junji Iwahara1& G. Marius Clore1
Kinetic data on a number of protein–protein associations have
provided evidence for the initial formation of a pre-equilibrium
encounter complex that subsequently relaxes to the final stereo-
specific complex1. Site-directed mutagenesis2–4and brownian
can be modulated by perturbations in charge distribution outside
the direct interaction surfaces. Furthermore, rate enhancement
through non-specific binding may occur by either a reduction in
dimensionality8or the presence of a short-range, non-specific
ment, we directly demonstrate the existence and visualize the
distribution of an ensemble of transient, non-specific encounter
tein–protein complex between the amino-terminal domain of
enzyme I and the phosphocarrier protein HPr. Neither the stereo-
specific complex10alone nor any single alternative conformation
can account fully for the intermolecular paramagnetic relaxation
enhancement data. Restrained rigid-body simulated annealing
enables us to obtain an atomic probability distribution map of the
lates with the electrostatic surface potentials on the interacting
proteins. Qualitatively similar results are presented for two other
The association of the N-terminal domain of enzyme I (EIN)
and the phosphocarrier protein HPr (dissociation constant,
ferase system11, is in fast exchange on the chemical shift scale10. The
structure of the stereospecific EIN–HPr complex has been solved by
NMR on the basis of nuclear Overhauser enhancement (NOE) and
residual dipolar coupling (RDC) data10. These data are fully consist-
ent with a single, unique conformation that readily accounts for the
phosphoryl transfer reaction between the two proteins10. However,
neither the NOE nor the RDCs are sensitive to the presence of low-
population (#10%) intermediates. To detect such intermediates, we
introduced a paramagnetic label at three sites on HPr, one at a time,
and measured the transverse paramagnetic relaxation enhancement
(PRE) rates, C2, of the backbone amide protons (1HN) of EIN. In a
fast exchanging system, the observed value of C2is the weighted
average of the C2values for the various states present in solution12,13.
Because C2is dependent on the sixth root of the distance (,r26.)
between the unpaired electron on the paramagnetic centre and the
observed proton, and because the C2rates at short distances are very
large owing to the large magnetic moment of the unpaired electrons,
low-population intermediates can be detected. Glu5, Glu25 and
Glu32 of HPr, which are all located outside the specific interaction
surface with EIN, were substituted individually by a cysteine residue,
which was then conjugated to EDTA through a disulphide linkage to
yield a (cysteaminyl-EDTA)-Cys adduct. The latter is chelated to
either Mn21(paramagnetic state) or Ca21(diamagnetic state)14.
These modifications do not change the net charge of HPr, nor do
they perturb the binding equilibrium with EIN.
The intramolecular1HN-C2rates for HPr in the EIN–HPR com-
plex are fully consistent with the static structure of HPr, with an
overall PRE Q-factor15(for all three paramagnetic sites combined)
of 0.18 and a correlation coefficient of 0.94 (Supplementary Fig. S1).
(The Q-factor is a quantitative measure of agreement between
observed and calculated C2rates and is given by equation (2) in
from the structure of the stereospecific complex as a function of
residue number reveals regions with large discrepancies (Fig. 1).
The correlation between observed and calculated intermolecular
1HN-C2rates is very poor, with an overall Q-factor of 0.61 (Fig. 2a).
In the stereospecific complex, the intermolecular contacts predomi-
nantly involve helices a2 and a29, the carboxy-terminal end of helix
a3 and the N-terminal end of helix a4 of the a-domain of EIN, and
helices a1 and a2 of HPr10. For each paramagnetic site, there are
predicted by the stereospecific complex (that is, they are in close
proximity to the paramagnetic labels): namely, residues 110–137,
50–92 and 73–140 for Glu5RCys, Glu25RCys and Glu32RCys,
respectively, within the a-domain of EIN (Fig. 1). However, there
($25A˚) from the paramagnetic labels but show large1HN-C2rates
that are inconsistent with the structure of the specific complex:
namely, residues 59–97, 105–124 and 20–71 for Glu5RCys,
are regions in the a/b domain, further away from the paramagnetic
centres, where the agreement is poor to moderate: namely, residues
23–37, 183–189 and 241–249 for Glu25RCys, and residues 184–189
and 232–249 for Glu32RCys (Fig. 1). The discrepancies cannot be
collisions between EIN and the paramagnetically labelled HPr
because, at the relatively low concentrations used (,300mM), no
significant PRE effects (.2s21) were observed for a control protein
(that does not interact with HPr) on addition of paramagnetically
labelled HPr. Thus, the observed intermolecular PRE data provide
unambiguous qualitative evidence for the existence of lowly popu-
lated (#10%) minor species in rapid exchange with the final stereo-
specific complex (see Supplementary Information).
A semi-quantitative depiction of the minor species was obtained
by using restrained rigid-body simulated annealing refinement18to
minimize the difference between observed and calculated1HN-C2
rates for all three paramagnetic sites simultaneously by representing
theminor non-specific states byanensembleofHPr molecules (with
reflects a population distribution). We carried out 100 independent
1Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0520, USA.
Vol 444|16 November 2006|doi:10.1038/nature05201
calculations with ensemble sizes N ranging from 1 to 20, varying the
percentage of the minor species (pminor) relative to the stereospecific
complex. Complete cross-validation19, leaving out random portions
of 10% of the complete PRE data set from all three sites, was done to
assess howwell thetestPRE datasets (10%excluded from therefine-
ment) are predicted by the working data sets (90% included in the
refinement). Introduction of a single minor species results in only a
modest decrease in the Q-factor, but, as the number of conformers is
increased, a large decrease in the Q-factor is observed (Fig. 2c).
formers required to satisfy the data is 10–20 (Fig. 2c) and that the
the minor species comprises many non-specific binding states that
reflect an ensemble of transient encounter complexes.
The Q-factor decreases rapidly as pminor is increased to 10%
(Fig. 2d), a value that is still consistent with the NOE and RDC data,
and thereafter slowly levels off. The Q-factor obtained by averaging
the1HN-C2rates over all ensembles (that is, the ensemble of ensem-
bles average, Qee) is systematically smaller than the average Q-factor
for the individual ensembles (Qe). This is due to the stochastic rather
than unique configuration of states within each ensemble, such that
averaging over all ensembles affords a better representation of the
data (Fig. 2c, d). For N520 and pminor510%, Qeehas a value of
0.21 and the correlation coefficient between observed and calculated
1HN-C2rates is 0.97 (Fig. 2b), which is comparable to the agreement
observed for the intramolecular PRE data (Supplementary Fig. S1).
The overall population of minor non-specific encounter complexes
may seem to be relatively high but is perhaps not unexpected for
a relatively weak protein–protein association. However, the occu-
pancy of any individual conformer in the ensemble of non-specific
characterization by any other experimental method.
The minor species were visualized by using a reweighted atomic
using an ensemble size of N520 (Fig. 3a–c, left). The distribution of
tially continuous but non-uniform. The probability density in the
region of the specific complex is low, indicating that alternative
modes of binding confined to the contact surfaces involved in the
stereospecific complex donot significantly contribute to thediscrep-
ancy between the observed PREs and those calculated on the basis of
the specific complex (Supplementary Figs S2 and S3).
Outside the specific interaction surface, the distribution of HPr
electrostatic potential isosurface21of EIN (Fig. 3a–c, right). EIN is an
acidic protein and large areas of its surface are swathed by a negative
electrostatic potential. About half the surface of HPr, including the
and the remaining surface (formed predominantly by the b-sheet) is
negatively charged. The HPr atomic probability density map heavily
located at the a/b domain are, on average, ,50A˚from HPr in the
stereospecific complex. In all cases, HPr preferentially uses its posi-
tively charged surface to interact with the negatively charged regions
of EIN (Supplementary Fig. S5c). Thus, the modifications used to
introduce paramagnetic groups on HPr do not affect the formation
of non-specific encounter complexes. These findings suggest that
the formation of non-specific EIN–HPr encounter complexes is
EIN residue number
E5C 2 (s–1)
E25C 2 (s–1)
E32C 2 (s–1)
∆ 2 (s–1)
Figure 1 | Observed and calculated intermolecular PREs for the EIN–HPr
complex. Shown is a comparison of observed intermolecular1HN-C2rates
(red diamonds) with those back-calculated (black lines) from the
one at a time at three sites (E5C, E25C and E32C) on HPr. Red crosses
indicate residues with1HN/15N cross-peaks that are broadened beyond
detection by PRE. Insets show the structure of the stereospecific EIN–HPr
complex with EIN colour coded according to the difference, DC2, between
the observed and calculated intermolecular1HN-C2rates for each
paramagnetic site. (HPr, green; three-member ensemble representation of
EDTA-Mn21with EDTA and linkage, orange, and Mn21, red spheres).
Population of minor species (%)
Q-factor = 0.61
Ensemble size N
Q-factor = 0.21
Figure 2 | Ensemble refinement and intermolecular PRE Q-factor.
a, b, Correlation between observed intermolecular1HN-C2rates (501 data
either alone (a) or with the addition of an ensemble of N520 to represent
the non-specific encounter complex (b; pminor510%; averaged over 100
and cross-validated (Q-free, green) Q-factors on ensemble size N
in c and d at N50 and pminor50, respectively, represent control
calculations in which pminor50 and the position of HPr for the single
specific complex is optimized by rigid-body simulated annealing to satisfy
the intermolecular PRE data. Error bars indicate the s.d.
NATURE|Vol 444|16 November 2006
predominantly driven by weak non-specific electrostatic attractions
between the two molecules.
The total solvent accessible surface area (ASA) buried at the inter-
faces of the non-specific encounter complexes is, on average, an
order of magnitude smaller than that of the stereospecific complex
(1,945A˚2; Fig. 3d). The non-specific interfaces are also much less
compact, with gap indices (defined as the ratio of gap volume to
buried interface ASA22) many times larger than that of the stereo-
specific complex (2.1A˚; Fig. 3d). In addition, the non-specific inter-
faces are more planar than the stereospecific one, indicative of the
absence of lock-and-key binding (Supplementary Table S1). These
observations are consistent with the correlation between the spatial
distribution of non-specific encounter complexes and electrostatic
potential isosurface, because electrostatic interactions are relatively
long range (with a 1/r distance dependence) and do not necessarily
reinforced by the observation that the interfacial composition of
charged residues is increased, whereas that of uncharged polar and
non-polar residues is decreased in the non-specific encounter com-
plexes relative to the stereospecific complex (Supplementary Fig.
Once a non-specific encounter complex is formed by weak non-
specific electrostatic interactions, HPr can carry out a two-dimen-
sional search on the surface of EIN, eventually falling into a narrow
energy funnel that leads directly to the stereospecific complex
characterized by an array of complementary van der Waals and
electrostatic interactions. The observation that the region on EIN
comprising the specific interaction surface for HPr is only minimally
occupied by non-specific encounter complexes (Fig. 3a) indicates
that once HPr reaches this region formation of the stereospecific
complex occurs with high probability.
The direct detection of non-specific encounter complexes by PRE
for two other weak (Kd<30–50mM), fast-exchanging protein–
protein complexes of the bacterial phosphotransferase system,
IIAMannitol–HPr (Fig. 4) and IIAMannose–HPr (Supplementary Fig.
S6), the stereospecific structures of which have been solved23,24.
With the paramagnetic label on HPr located at Glu5RCys, on the
opposite face to the stereospecific binding site, there are regions in
both complexes where the observed intermolecular1HN-C2rates are
much larger than those back-calculated from the structures of the
stereospecific complexes. The largest discrepancies involve acidic
residues of IIAMannitoland IIAMannose, the distribution of which cor-
relates qualitatively with the negative electrostatic potential on the
surface of these proteins. Thus, in all likelihood the observations
Buried ASA at interface (Å2) Gap index (Å)
0 20406080 10008001600
Figure 3 | Characterization of non-specific EIN–HPr encounter complexes.
a–c, Left, overall distribution of HPr molecules obtained from 100
calculations (N520) displayed as a reweighted atomic probability density
map20(plotted at a threshold of 20% maximum, green) on the molecular
surface of EIN (colour coded by electrostatic potential, 68kT). Right,
EIN, calculated at 65kT, displayed as red (negative) and blue (positive)
of N520 is shown in the right panel of a. The location of HPr in the
stereospecificcomplexisshownas a blueribbonin all panels. d, Histograms
of interface-buried ASA and gap index for the non-specific encounter
complexes. Red lines indicate values for the stereospecific complex.
IIAMannitol residue number
E5C 2 (s–1)
Q-factor = 0.89
∆ 2 (s–1)
Figure 4 | Observed and calculated PRE1HN-C2values for the
IIAMannitol–HPr complex. EDTA-Mn21paramagnetic labels were
introduced onto HPr at E5C. a, Comparison of observed intermolecular
1HN-C2rates (red diamonds) with those back-calculated (black lines) from
the stereospecific complex. Red crosses indicate residues with1HN/15N
the s.d. b, Structure of the stereospecific complex with IIAMannitolcolour
coded according to the difference, DC2, between observed and calculated
complex. Residues of IIAMannitolthat show large DC2are coloured cyan.
d, Electrostatic potential isosurface of IIAMannitol(65kT in blue and red,
respectively). In c and d, HPr is shown as a green ribbon. The same view is
shown in b–d.
NATURE|Vol 444|16 November 2006
reported herereflect ageneral phenomenon ofprotein–protein asso- Download full-text
ciation in which the initial formation of non-specific encounter
complexes through long-range electrostatic interactions (possibly
supplemented by short-range van der Waals interactions) facilitates
the rapid formation of a stereospecific complex by reducing the
dimensionality of the search process.
Sample preparation and NMR spectroscopy. Details of sample preparation are
provided in Supplementary Information. We acquired PRE data on a Bruker
DRX-600 spectrometer as described15(see Supplementary Information).
from the structures of the stereospecific complexes by using a three-conformer
ensemble representation for the EDTA-Mn21groups to account for their flex-
ibility (see Supplementary Information)15.
Ensemble refinement against intermolecular PREs. Refinement against the
intermolecular PREs for the EIN–HPr complex was carried out by rigid body
refinement with Xplor-NIH18subject to a target function comprising the PRE
data for all three paramagnetic sites15, a quartic van der Waals repulsion term25
(to prevent atomic overlap between EIN and HPr) and a very weak radius of
gyration restraint26(to ensure that each member of the ensemble makes at least
some intermolecular contacts). Calculations were carried out either by keeping
EIN fixed and allowing an ensemble of HPr molecules to rotate and translate, or
by the converse (HPr fixed and an ensemble of EIN molecules), with essentially
identical results (Supplementary Fig. S5). Further details of the calculations are
provided in Supplementary Information. The calculated PRE for residue i,
C2calc(i), is given by
where l is the fraction of the stereospecific complex (l512pminor), N is the
C2rate for residue i in the stereospecific complex, and C2non-specific(i,j) is the
calculated C2rate for residue i of member j of the non-specific encounter com-
plex ensemble. The PRE Q-factor is a measure of the agreement between
observed and calculated values of C2and is given by:
where C2obs(i) is the observed C2rate for residue i. Two Q-factors are reported:
Qeis the average Q-factor ,Q. for all calculated n ensembles, with C2calc(i)
computed by equation (1); Qeeis the ensemble of ensembles average Q-factor,
computed by using the average value of C2calc(i) over all n ensembles, with
C2calc(i) given by:
Analysisofcomplexes.Electrostaticpotentials were calculated with APBS21and
are displayed in PyMol27. Solvent ASA buried at the interface and gap volume
were calculated with Xplor-NIH18and SURFNET28, respectively.
Received 23 May; accepted 4 September 2006.
Published online 15 October 2006.
and Protein Folding (Freeman & Co, New York, 1999).
Schreiber, G. & Fersht, A. R. Rapid electrostatically assisted association of
proteins. Nature Struct. Biol. 3, 427–431 (1996).
Vijaykumar, M. et al. Electrostatic enhancement of diffusion-controlled
protein–protein association: comparison of theory and experiment on barnase
and barstar. J. Mol. Biol. 278, 1015–1024 (1998).
Selzer, T., Albeck, S. & Schreiber, G. Rational design of faster associating and
tighter binding protein complexes. Nature Struct. Biol. 7, 537–541 (2000).
Northrup, S.H.,Boles, J. O. &Reynolds, J. C.L.Brownian dynamics ofcytochrome
c and cytochrome c peroxidase association. Science 241, 67–70 (1988).
Gabdoulline, R. R. & Wade, R. C. Biomolecular diffusional association. Curr. Opin.
Struct. Biol. 12, 204–213 (2002).
7. Spaar, A., Dammer, C., Gabdoulline, R. R., Wade, R. C. & Helms, V. Diffusional
encounter of barnase and barstar. Biophys. J. 90, 1913–1924 (2006).
Adams, G. & Debruck, M. in Structural Chemistry and Molecular Biology (eds Rich,
A. & Davidson, N.) 198–215 (Freeman & Co, San Francisco, CA, 1968).
Zhou, H-X. & Szabo, A. Enhancement of association rates by nonspecific binding
to DNA and cell membranes. Phys. Rev. Lett. 93, 178101 (2004).
10. Garrett, D. S., Seok, Y-J., Peterkofsky, A., Gronenborn, A. M. & Clore, G. M.
Solution structure of the 40,000 Mrphosphoryl transfer complex between
the N-terminal domain of enzyme I and HPr. Nature Struct. Biol. 6, 166–173
11. Postma, P. W., Lengeler, J. W. & Jacobson, G. R. in Escherichia coli and Salmonella:
12. Iwahara, J. & Clore, G. M. Detecting transient intermediates in macromolecular
binding by paramagnetic NMR. Nature 440, 1227–1230 (2006).
13. Iwahara, J., Schwieters, C. D. & Clore, G. M. Characterization of nonspecific
protein–DNA interactions by1H paramagnetic relaxation enhancement. J. Am.
Chem. Soc. 126, 12800–12808 (2004).
14. Ebright, Y. W., Chen, Y., Pendergrast, P. S. & Ebright, R. H. Incorporation of an
EDTA–metal complex at a rationally selected site within a protein: application to
EDTA–iron DNA affinity cleaving with catabolite gene activator protein (CAP)
and Cro. Biochemistry 31, 10664–10670 (1992).
15. Iwahara, J., Schwieters, C. D. & Clore, G. M. Ensemble approach for NMR
structure refinement against1H paramagnetic relaxation enhancement data
arising from a flexible paramagnetic group attached to a macromolecules. J. Am.
Chem. Soc. 126, 5879–5896 (2004).
16. Donaldson, L. W. et al. Structural characterization of proteins with an attached
ATCUN motif by paramagnetic relaxation enhancement NMR spectroscopy. J.
Am. Chem. Soc. 123, 9843–9847 (2001).
17. Pintacuda, G. & Otting, G. Identification of protein surfaces by NMR
measurements with a paramagnetic Gd(III) chelate. J. Am. Chem. Soc. 124,
18. Schwieters, C. D., Kuszewski, J. J. & Clore, G. M. Using Xplor-NIH for NMR
molecular structure determination. Prog. NMR Spectrosc. 48, 47–62 (2006).
19. Bru ¨nger, A. T., Clore, G. M., Gronenborn, A. M. &Nilges, M. Assessing the quality
of solution nuclear magnetic resonance structures by complete cross-validation.
Science 261, 328–331 (1993).
20. Schwieters, C. D. & Clore, G. M. Reweighted atomic densities to represent
ensembles of NMR structures. J. Biomol. NMR 23, 221–225 (2002).
21. Baker, N. A., Sept, D., Joseph, S., Holst, M. J. & McCammon, J. A. Electrostatics of
nanosystems: application to microtubules and the ribosome. Proc. Natl Acad. Sci.
USA 98, 10037–10041 (2001).
22. Jones, S. & Thornton, J. M. principles of protein–protein interactions. Proc. Natl
Acad. Sci. USA 93, 13–20 (1996).
23. Cornilescu, G. et al. Solution structure of the phosphoryl transfer complex
between the cytoplasmic A domain of the mannitol transporter IIMannitoland HPr
of the Eschrichia coli phosphotransferase system. J. Biol. Chem. 277,
24. Williams, D. C., Cai, M., Suh, Y-J., Peterkofsky, A. & Clore, G. M. Solution NMR
structure of the 48 kDa IIAMannose–HPr complex of the Escherichia coli mannose
phosphotransferase system. J. Biol. Chem. 280, 20775–20784 (2005).
25. Nilges, M., Gronenborn, A. M., Bru ¨nger, A. T. & Clore, G. M. Determination of
three-dimensional structures ofproteins bysimulatedannealing with interproton
barley serine proteinase inhibitor 2. Protein Eng. 2, 27–38 (1988).
26. Kuszewski, J., Gronenborn, A. M. & Clore, G. M. Improving the packing and
accuracy of NMR structures with a pseudopotential for the radius of gyration. J.
Am. Chem. Soc. 121, 2337–2338 (1999).
27. DeLano, W. L. The PyMOL Molecular Graphics System (DeLano Scientific, San
Carlos, CA, USA, 2002).
28. Laskowski, R. A. SURFNET: a program for visualizing molecular surfaces, cavities
and intermolecular interactions. J. Mol. Graph. 13, 323–330 (1995).
Supplementary Information is linked to the online version of the paper at
Acknowledgements We thank C. Schwieters, A. Szabo and C. Bewley for
discussions; and C. Byeon for assistance with initial sample preparation. This work
was supported by funds from the Intramural Program of the NIH, NIDDK and the
AIDS Targeted Antiviral program of the Office of the Director of the NIH (to
Author Information Reprints and permissions information is available at
www.nature.com/reprints. The authors declare no competing financial interests.
Correspondence and requests for materials should be addressed to G.M.C.
NATURE|Vol 444|16 November 2006