Membrane Protein Structure

Calculations from experimental solid-

state NMR data

1

Recent Developments at the BTRC.

Simultaneous Assignment and Structure Refinement.

Tian et al. (2012) J Magn Reson 214:42.

Membrane protein structure determination:

human chemokine receptor CXCR1 in liposomes

Marassi et al. (2011) Methods 55:363.

Das et al. (2012) J Am Chem Soc 134:2047.

Park et al. (2012) Nature 491:779.

Improved CS prediction by conformational sampling.

Tian et al. (2012) J Biomol NMR 54:237.

Protein structures in the PDB.

INPUT

protein amino acid sequence

experimental restraints

(CS, DC, CSA, PRE, distances)

STARTING MODEL

coarse-grained Rosetta model

(all-atom implicit membrane potential)

REFINE

NMR restrained MD (XPLOR-NIH)

VALIDATE

against all experimental data

Membrane protein structure calculation from solid-state NMR.

human CXCR1 in liposomes

(PDB 2LNL)

Marassi et al. (2011) Methods 55:363.

Das et al. (2012) J Am Chem Soc 134:2047.

Park et al. (2012) Nature 491:779.

Improve chemical shift prediction by conformational sampling.

• Predict structural models and select ensemble based on energy and RMSD.

• Predict CS for each model in structural ensemble (ShiftX, SPARTA+, …).

• Average CS over all structures.

• Compare to experimental CS.

• CS reflect time-average of conformational

states of the protein.

• Prediction accuracy is improved by

averaging over ensemble of structures

predicted from sequence (i.e. with Rosetta).

• Potential for guiding NMR resonance

assignment.

Bacterial MerFt (PDB 2LJ2)

Tian et al. (2012) J Biomol NMR 54:237.

Method for Simultaneous Assignment & Structure Refinement.

• AssignFit module developed for XPLOR-NIH.

• AssignFit example scripts and data files released with XPLOR-NIH 2.29.

fd coat protein in planar bilayers (PDB 2MZT)

Tian et al. (2012) J Magn Reson 214:42.

Detection and analysis of dynamics from orientation restraints

Teriete et al. (2007) Biochemistry 46:6774.

human FXYD1 in micelles. fd coat protein in phospholipid bilayers

Effective Energy Function / Implicit Membrane Model

(EEFx / IMMx) for XPLOR-NIH.

Energy Terms for NMR-restrained structure calculations

Experimental restraints

CDIH + NOE + PRE + DC + CSA …

Database / empirical restraints

torsionDBPot + RAMA + Rgyr …

Molecular interaction terms

BOND + ANGL + IMPR + DIHE +

VDWREPEL standard NMR calculations

VDWLJ + ELEC + SLV EEFx

VDWLJ + ELEC + SLV + IMM EEFx / IMMx

ETOTAL

EEXPERIMENTAL

+

EKNOWLEDGE

+

ESYSTEM

Clore & Gronenborn (1989) Crit Rev Biochem Mol Biol 24:479.

Lazaridis & Karplus (1999) Proteins 35:133.

Lazaridis (1999) Proteins 52:176.

Tian et al. (2014) Submitted.

Release with XPLOR-NIH 2.36.

EEFx Effective Energy Function sustains native protein structures.

• Unrestrained MD simulations (1 ns, 300 K, real atomic masses, XPLOR-NIH).

• EEFx outperforms vacuum, VDWLJ and VDWREPEL force fields.

Staph. protein A Z-domain

(PDB: 1Q2N)

EEFx Effective Energy Function sustains native protein structures.

•EEFx improves structural accuracy.

•EEFx improves RDC cross validation.

•EEFx optimizes H bonds.

•EEFx optimizes packing.

• Unrestrained MD simulations (100 ps, 300 K, real atomic masses, XPLOR-NIH).

• 11 test proteins with varying sizes (60-260 residues) and varying αβ folds.

EEFx Effective Energy Function recognizes native fold.

• EEFx and Rosetta funnel towards native structure.

• EEFx provides wider dynamic range for discrimination of folded states.

Structures predicted with Rosetta and scored by Rosetta or EEFx energy.

Protein G, B1 domain (PDB: 3GB1)

EEFx Effective Energy Function improves NMR structures:

effect on calculations with sparse restraints.

• Calculate structures from extended templates with simulated annealing.

• Restrain with dihedral angles and limited NOE distances (short-range NOEs removed; long-

range NOEs randomly eliminated from full data set).

•EEFx directs calculation towards native fold.

•EEFx improves structural accuracy.

•EEFx improves structural precision.

• Calculate structures from extended templates with simulated annealing.

• Restrain with dihedral angles and all available NOE distances.

• Cross validate with RDCs.

•EEFx maintains agreement with other experimental

and conformational energy terms.

•EEFx improves structural quality.

•EEFx compares well with Water-refine.

•EEFx is computationally efficient: calculations are only

2.5 times longer than with REPEL.

EEFx Effective Energy Function improves NMR structures:

effect on structural quality.

Introducing the membrane environment:

IMMx Implicit Membrane Model

• Model membrane environment based on known profiles (X-ray and neutron diffraction).

• Distance-dependent electrostatic screening and solvation.

fd coat protein (PDB: 2MZT)