Local Ordering at the N–H Sites of the Rho GTPase Binding Domain of Plexin-B1: Impact of Dimerization

Abstract

We have developed a new molecular dynamics (MD) based method for describing analytically local potentials at mobile N–H sites in proteins. Here we apply it to the monomer and dimer of the Rho GTPase binding domain (RBD) of the transmembrane receptor plexin-B1 to gain insight into dimerization, which can compete with Rho GTPase binding. In our method, the local potential is given by linear combinations, u (D L,K ), of the real combinations of the Wigner rotation matrix elements, D L,K , with L = 1–4 and appropriate symmetry. The combination that “fits best” the corresponding MD potential of mean force, u (MD), is the potential we are seeking, u (D L,K – BEST). For practical reasons the fitting process involves probability distributions, P eq ∝ exp­(−u), instead of potentials, u. The symmetry of the potential, u (D L,K ), may be related to the irreducible representations of the D 2h point group. The monomer (dimer) potentials have mostly Ag and B2u (B1u and B2u) symmetry. For the monomer, the associated probability distributions are generally dispersed in space, shallow, and centered at the “reference N–H orientation” (defined in section 3.1. below); for the dimer many are more concentrated, deep and centered away from the “reference N–H orientation”. The u (D L,K ) functions provide a consistent description of the potential energy landscape at protein N–H sites. The L1-loop of the plexin-B1 RBD is not seen in the crystal structure, and many resonances of the L4 loop are missing in the NMR 15N–1H HSQC spectrum of the dimer; we suggest reasons for these features. An allosteric signal transmission pathway was reported previously for the monomer. We find that it has shallow N–H potentials at its ends, which become deeper as one proceeds toward the middle, complementing structurally the previously derived dynamic picture. Prospects of this study include correlating u (D L,K – BEST) with MD force-fields, and using them without further adjustment in NMR relaxation analysis schemes.

Conformational Entropy from Restricted Bond-Vector Motion in Proteins: The Symmetry of the Local Restrictions and Relation to NMR Relaxation

“… 16 , 19 In accordance with typical experimental data, the lowest even L terms, and in some cases the lowest odd L terms, have been kept, yielding u = – c 0 2 D 00 2 – c 2 2 ( D 02 2 + D 0–2 2 ) for nonpolar ordering 17 , 18 , 20 − 22 and u = −c 0 1 D 00 1 – c 1 1 ( D 0–1 1 – D 01 1 ) for polar ordering. 23 − 26 …”

“…Moreover, using such optimized potentials, new insights into the dimerization of the Rho GTPase binding domain of plexin-B1 (in brief, plexin-B1 RBD) were gained. 26 In future work, we plan to incorporate these potentials unchanged into SRLS data-fitting schemes. This will improve the picture of structural dynamics to be obtained due to better potentials, and better characterization of this picture as additional parameters can now be determined with data fitting.…”

“…In recent years, we developed the two-body coupled-rotator slowly relaxing local structure (SRLS) approach − for the analysis of NMR relaxation in proteins. − In SRLS, the local potential is expanded in the basis set of the real linear combinations of the Wigner rotation matrix elements, D 0 K L (in brief, real Wigner functions). , In accordance with typical experimental data, the lowest even L terms, and in some cases the lowest odd L terms, have been kept, yielding u = – c 0 2 D 00 2 – c 2 2 ( D 02 2 + D 0–2 2 ) for nonpolar ordering ,,− and u = −c 0 1 D 00 1 – c 1 1 ( D 0–1 1 – D 01 1 ) for polar ordering. − …”

“…In practice, this is often hindered by limited experimental data. We pursued the idea of potential enhancement outside of the scope of SRLS , as follows. Linear combinations of real Wigner functions with L = 1–4 were created and optimized against the corresponding potential of mean force (POMF) obtained with MD simulations.…”

“…Comparison between the best-fit Wigner function and the POMF indicated that the set of terms with L = 1–4 suffices for obtaining good agreement. Moreover, using such optimized potentials, new insights into the dimerization of the Rho GTPase binding domain of plexin-B1 (in brief, plexin-B1 RBD) were gained . In future work, we plan to incorporate these potentials unchanged into SRLS data-fitting schemes.…”