2.3 Backbone Dynamics of L-hIGFBP2 from 15N
relaxation
Insight into the functional regulation of IGF-1 by L-h IGFBP2 was
achieved by studying the dynamics of L-h IGFBP2 in the free and
IGF-1-bound forms using 15N relaxation. Reduced
spectral density mapping was used to examine dynamics in the
microsecond-millisecond (µs-ms) and picosecond-nanosecond (ps-ns)
regimes. The 15N relaxation rates
(R1, R2, R1ρ) and15N-1H heteronuclear nuclear
Overhauser effects (HetNOE) were measured at a 1H
resonance frequency of 800 MHz (Fig. 4 ). Based on15N R1 and R1ρrelaxation values, an average overall rotational correlation time of
~3 ns for the disordered linker domain was obtained for
unbound L-h IGFBP2. The average overall correlation time for the
linker domain in full-length h IGFBP2 (32 kDa) determined by a
similar method was ~4 ns at 293 K, implying that the
disordered linker domain retains a high degree of flexibility in the
full-length form, largely unaffected by the presence of the N- and
C-domains.
The 15N R1, R2, and15N-1H het-NOE values and a plot of
the calculated spectral density functions- J (0),J (ωN), and J (0.87*ωH) for
L-h IGFBP2 in free and IGF-1-bound forms are shown in Fig.
4 and Fig. 5 , respectively. Several important observations can
be made. First, L-h IGFBP2 exhibits a high degree of flexibility
in both the free and IGF-1-bound forms, as reflected by theJ (0.87*ωH) and J (0) values, with the
latter being significantly less than 2/5τc for most
residues (where τc is the rotational correlation time of
a rigid isotropically tumbling protein of equivalent size). Second, the15N relaxation rates for residues K150-E161, Q165, and
M166 of the linker domain are significantly perturbed by IGF-1 in
complex with L-h IGFBP2, as the large complex causes a great
increase in correlation time (τc ) which in turn
causes the fast T2 relaxation (Fig. 4) . In the
full-length h IGFBP2 complex the intensities of cross-peaks
corresponding to these residues in the 2D [15N,1H] HSQC spectrum are reduced owing to the formation
of a large complex which causes an increase in correlation time
(τc ), resulting in the fast T2relaxation and increased NMR line-width (Fig. S6 ). Third, largeJ (0) values indicative of dynamics in the µs-ms regime are
significantly enhanced for L-domain residues involved in binding IGF-1
(K150-E161), as well as those distant from the binding site (V110, N113,
H117, H172, Q165, M166, L174, and L182). This increase in J (0)
values can be attributed to the larger size of the complex and slow
conformational exchange in the µs-ms regime, and is quantified by the
exchange rate, Rex, which was estimated by measuring the15N transverse relaxation rate in the rotating frame
(R1ρ) at 800 MHz for both the unbound and bound forms of
L-h IGFBP2. Values of Rex calculated using the
difference in J (0) values obtained with R2 and
R1ρ are plotted in Fig. 5 . Notably, an
overall increase in Rex is observed in the IGF-bound
complex for residues of L-h IGFBP2 both close to and distant from
the binding site. This implies that in the IGF-1-bound complex
L-h IGFBP2 populates an ensemble of alternate conformations that
interconvert on the µs-ms timescale. The region bound to IGF-1 exhibits
a ‘reduced’ level of conformational dynamics, such that some of the
ns-ps timescales have now entered the µs-ms timescale (and therefore now
entered the exchange regime in these experiments). Interestingly, it is
known that proteolytic cleavage of h IGFBP2 by the
pregnancy-associated plasma protein-A (PAPP-A) is enhanced in the
IGF-1-bound state. As shown in Fig. 7 , the region K150-E161
from the disordered linker domain of h IGFBP2 has a helical
propensity, as predicted by AlphaFold; this binds to IGF-1 and shows a
change in exchange in µs-ms timescale due to the formation of the larger
complex upon binding. The IGF-1 bound complex has a higher correlation
time (τc ) which in turn causes the fast
T2 relaxation, resulting in a higher relaxation rate, as
the relaxation times and relaxation rates are simple inverses of each
other. The majority of the L-h IGFBP2 residues recognized by
proteases (Fig. 3 ) show increases in
Rex upon IGF binding (Fig. 5 ). This explains
the IGF-dependent dynamic modulation of a protease cleavage site region
in the intrinsically disordered linker domain of h IGFBP2.
To estimate the conformational entropy associated with binding IGF-1,
approximate backbone NH order parameters (S 2)
using J (0) and J (ωN) were calculated for
L-h IGFBP2. J (0) and J (ωN)
calculated from R1ρ were used to avoid the effect of
conformational exchange when estimating S 2values. Order parameters were calculated for both free and bound forms
of L-h IGFBP2 and the change in conformational entropy (ΔS) was
estimated using the calculated S 2 values (for
residues with S 2 < 1) (Eq. 2;
Fig. 6 ). The overall ΔS value (summed over all residues) of
~100 J/mole (0.024 kcal/mol) implies an increase in
entropy for the system upon IGF-1 binding. The contribution of the
conformational entropy to the free energy of binding is given by -TΔS,
which yields a contribution of -7 kcal/mol.
Discussion
In recent years, the concept of “fuzzy complexes” in IDPs has been
described , which proposes that functionally important regions of IDPs
in protein complexes can retain their structural disorder. In fuzzy
complexes, dynamic regulation ensues when the ensemble average
population of conformers of the IDP and/or their flexibility are
affected upon ligand binding. The current study involving the
intrinsically disordered linker domain of human IGFBP2 exemplifies such
a case.
In the IGF system, proteolysis plays a crucial role in regulating the
bioavailability of IGFs. IGFBP levels are regulated by proteolysis
following their secretion from the cell and the resulting proteolytic
fragments have reduced affinity for IGF ligands. The net effect is an
increase in IGFs availability for interaction with the IGF-1R. Thus,
efforts to reduce protease action could have a beneficial effect on
reducing IGF-1R activity in cancer. Proteolysis of IGFBPs has been
observed to be both IGF-dependent and IGF-independent; IGF-dependent
proteolysis has been observed for IGFBP2 and in IGFBP4, whereas IGFBP3
and IGFBP5 undergo IGF-independent proteolysis. K150-E161 residues in
IGFBP2 are more ordered (less disorder disposition, Fig. S4 ),
and our experimental results show an increase in dynamics for those
residues in IGFBP2 after binding with IGF-1. As these K150-E161 residues
in the linker region become more flexible after binding, the
conformational ensemble populated by the linker domain of IGFBP2 shifts
so that it is more readily recognized by the protease and/or is more
amenable to proteolysis. This brings an interesting link for why binding
to IGF-1 can be so important for proteolysis for h IGFBP2, which
is not the case for IGFBP3 and IGFBP5. An example of an IGF-dependent
protease action on h IGFBP2 is PAPP- A, which cleavesh IGFBP2 in an IGF-dependent manner at a single site between
Gln165 and Met166, to yield two proteolytic fragments having weak IGF
binding affinity.
The different susceptibilities of the different IGFBPs to proteolysis
have been attributed to ligand-induced conformational changes. Our
studies demonstrate that K150-E161, Q165, and M166 residues of
L-h IGFBP2, which are involved in binding IGF-1, exhibit enhanced
conformational exchange upon IGF binding (Fig. 4 ). Moreover,
this enhanced conformational exchange is not confined to the binding
site but extends to some distant residues (V110, N113, H117, H172, Q165,
M166, L174, and L182) (Fig. 4 ) and is accompanied by an
increase in conformational entropy. This implies distant dynamic
regulation, where changes in protein dynamics induced by ligand binding
extend to residues distant from the ligand-binding site, even in the
absence of a well-defined conformational change. The increase in µs-ms
motions of residues in the vicinity of protease cleavage sites of
L-h IGFBP2 provides an interesting link to proteolytic cleavage
upon binding IGF-1 as it is well known that changes in conformational
dynamics upon ligand binding are important for regulation of
proteolysis. In the presence of IGF-1, the conformational ensemble
populated by the linker domain of IGFBP2 shifts so that it is more
readily recognized by the protease and/or is more amenable to
proteolysis. Considering the current findings, we therefore propose that
dynamic regulation in the linker domain of IGFBP2 plays an important
role in its susceptibility to PAPP-A proteolytic cleavage.
These results have significant implications for the development of
IGFBPs (mutants and/or chimeras) as antagonists of IGF-1R activation
that can block IGF-1R mediated tumor progression. Most current cancer
therapeutics target the IGF-signaling pathway and focus on blocking the
IGF-1R directly (kinase inhibitors) and/or its downstream effectors.
However, a drawback of this approach is the resulting high serum IGF-1
levels in response to targeted inhibition of IGF-1R and adverse
side-effects and/or toxicities arising from potential interference with
the insulin pathway. It has been suggested recently that therapeutics
targeting the interaction of IGFs with IGFBPs may overcome these serious
drawbacks. For example, IGFBPs engineered to be protease–resistant by
mutating or deleting the protease cleavage sites in the linker domain
should act as IGF antagonists. Recently, in separate studies, engineered
protease-resistant h IGFBP2 and h IGFBP4 were found to
inhibit tumor growth in breast cancer. Interestingly, the engineered
protease-resistant form of h IGFBP2 lacking residues 114-170
(des(114-170)) retains high-affinity binding to IGF-1 and IGF-2, with
only a 1.6-2-fold reduction in affinity compared to full-lengthh IGFBP2. The present study may now explain the loss in binding
affinity of des(114-170) towards both IGFs compared to the full-length
protein by the fact that residues K150-E161 of the linker domain, which
facilitate IGF binding (Fig. S7 ), were deleted from the
construct. This suggests that, in addition to alteration of the protease
cleavage sites, more potent IGFBP-based antagonists could be designed by
considering the binding affinity of the linker domain for the IGFs and
taking into account the resulting change in dynamics upon binding. These
studies will facilitate the development of future IGFBP-based
antagonists.
In summary, our studies of the intrinsically disordered linker domain of
human IGFBP2 provide new insights into the regulatory mechanisms in the
IGF system. Contrary to currently held models, the intrinsically
disordered linker domain of IGFBP2 is involved in binding IGF-1.
L-h IGFBP2 does not undergo a well-defined conformational change
upon binding its ligand, but binding is accompanied by a significant
change in dynamics on both the millisecond-microsecond and
picosecond-nanosecond time scales. This is an example of functional
regulation in an intrinsically disordered protein complex by dynamic
regulation, which is being recognized increasingly in recent years.
Materials and Methods