Microsecond Backbone Motions Modulate the Oligomerization of the DNAJB6 Chaperone

DNAJB6 is a prime example of an anti-aggregation chaperone that functions as an oligomer. DNAJB6 oligomers are dynamic and subunit exchange is critical for inhibiting client protein aggregation. The T193A mutation in the C-terminal domain (CTD) of DNAJB6 reduces both chaperone self-oligomerization and anti-aggregation of client proteins, and has recently been linked to Parkinson's disease. Here, we show by NMR, including relaxation-based methods, that the T193A mutation has minimal effects on the structure of the β-stranded CTD but increases the population and rate of formation of a partially folded state. The results can be rationalized in terms of β-strand peptide plane flips that occur on a timescale of ≈100 μs and lead to global changes in the overall pleat/flatness of the CTD, thereby altering its ability to oligomerize. These findings help forge a link between chaperone dynamics, oligomerization and anti-aggregation activity which may possibly lead to new therapeutic avenues tuned to target specific substrates

Structural Basis of the Association of HIV-1 Matrix Protein with DNA

HIV-1 matrix (MA) is a multifunctional protein that is synthesized as a polyprotein that is cleaved by protease during viral maturation. MA contains a cluster of basic residues whose role is controversial. Proposed functions include membrane anchoring, facilitating viral assembly, and directing nuclear import of the viral DNA. Since MA has been reported to be a component of the preintegration complex (PIC), we have used NMR to probe its interaction with other PIC components. We show that MA interacts with DNA and this is likely sufficient to account for its association with the PIC

Visualizing spatially correlated dynamics that directs RNA conformational transitions

RNAs fold into three- dimensional ( 3D) structures that subsequently undergo large, functionally important, conformational transitions in response to a variety of cellular signals(1-3). RNA structures are believed to encode spatially tuned flexibility that can direct transitions along specific conformational pathways(4,5). However, this hypothesis has proved difficult to examine directly because atomic movements in complex biomolecules cannot be visualized in 3D by using current experimental methods. Here we report the successful implementation of a strategy using NMR that has allowed us to visualize, with complete 3D rotational sensitivity, the dynamics between two RNA helices that are linked by a functionally important trinucleotide bulge over timescales extending up to milliseconds. The key to our approach is to anchor NMR frames of reference onto each helix and thereby directly measure their dynamics, one relative to the other, using 'relativistic' sets of residual dipolar couplings ( RDCs)(6,7). Using this approach, we uncovered super- large amplitude helix motions that trace out a surprisingly structured and spatially correlated 3D dynamic trajectory. The two helices twist around their individual axes by approximately 536 and 1106 in a highly correlated manner ( R = 0.97) while simultaneously ( R = 0.81 - 0.92) bending by about 94 degrees. Remarkably, the 3D dynamic trajectory is dotted at various positions by seven distinct ligand- bound conformations of the RNA. Thus even partly unstructured RNAs can undergo structured dynamics that directs ligand- induced transitions along specific predefined conformational pathways.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/62506/1/nature06389.pd

A unifying probabilistic framework for analyzing residual dipolar couplings

Residual dipolar couplings provide complementary information to the nuclear Overhauser effect measurements that are traditionally used in biomolecular structure determination by NMR. In a de novo structure determination, however, lack of knowledge about the degree and orientation of molecular alignment complicates the analysis of dipolar coupling data. We present a probabilistic framework for analyzing residual dipolar couplings and demonstrate that it is possible to estimate the atomic coordinates, the complete molecular alignment tensor, and the error of the couplings simultaneously. As a by-product, we also obtain estimates of the uncertainty in the coordinates and the alignment tensor. We show that our approach encompasses existing methods for determining the alignment tensor as special cases, including least squares estimation, histogram fitting, and elimination of an explicit alignment tensor in the restraint energy

Mapping the encounter state of a transient protein complex by PRE NMR spectroscopy

Many biomolecular interactions proceed via a short-lived encounter state, consisting of multiple, lowly-populated species invisible to most experimental techniques. Recent development of paramagnetic relaxation enhancement (PRE) nuclear magnetic resonance (NMR) spectroscopy has allowed to directly visualize such transient intermediates in a number of protein-protein and protein-DNA complexes. Here we present an analysis of the recently published PRE NMR data for a protein complex of yeast cytochrome c (Cc) and cytochrome c peroxidase (CcP). First, we describe a simple, general method to map out the spatial and temporal distributions of binding geometries constituting the Cc-CcP encounter state. We show that the spatiotemporal mapping provides a reliable estimate of the experimental coverage and, at higher coverage levels, allows to delineate the conformational space sampled by the minor species. To further refine the encounter state, we performed PRE-based ensemble simulations. The generated solutions reproduce well the experimental data and lie within the allowed regions of the encounter maps, confirming the validity of the mapping approach. The refined encounter ensembles are distributed predominantly in a region encompassing the dominant form of the complex, providing experimental proof for the results of classical theoretical simulations

NMRDyn: A Program for NMR Relaxation Studies of Protein Association

Self-association is an important biological phenomenon that is associated with many cellular processes. NMR relaxation measurements provide data about protein molecular dynamics at the atomic level and are sensitive to changes induced by self-association. Thus, measurements and analysis of NMR relaxation data can provide structurally resolved information on self-association that would not be accessible otherwise. Here, we present a computer program, NMRdyn, which analyses relaxation data to provide parameters defining protein self-association. Unlike existing relaxation analysis software, NMRdyn can explicitly model the monomer-oligomer equilibrium while fitting measured relaxation data. Additionally, the program is packaged with a user-friendly interface, which is important because relaxation data can often be large and complex. NMRdyn is available from http://research1t.imb.uq.edu.au/nmr/NMRdyn

Near-complete backbone resonance assignments of acid-denatured human cytochrome c in dimethylsulfoxide: a prelude to studying interactions with phospholipids

Human cytochrome c plays a central role in the mitochondrial electron transfer chain and in the intrinsic apoptosis pathway. Through the interaction with the phospholipid cardiolipin, cytochrome c triggers release of pro-apoptotic factors, including itself, from the mitochondrion into the cytosol of cells undergoing apoptosis. The cytochrome c/cardiolipin complex has been extensively studied through various spectroscopies, most recently with high-field solution and solid-state NMR spectroscopies, but there is no agreement between the various studies on key structural features of cytochrome c in its complex with cardiolipin. In the present study, we report backbone 1H, 13C, 15N resonance assignments of acid-denatured human cytochrome c in the aprotic solvent dimethylsulfoxide. These have led to the assignment of a reference 2D 1H-15N HSQC spectrum in which out of the 99 non-proline residues 87% of the backbone amides are assigned. These assignments are being used in an interrupted H/D exchange strategy to map the binding site of cardiolipin on human cytochrome c

Diverging Mechanisms of Activation of Chemokine Receptors Revealed by Novel Chemokine Agonists

CXCL8/interleukin-8 is a pro-inflammatory chemokine that triggers pleiotropic responses, including inflammation, angiogenesis, wound healing and tumorigenesis. We engineered the first selective CXCR1 agonists on the basis of residue substitutions in the conserved ELR triad and CXC motif of CXCL8. Our data reveal that the molecular mechanisms of activation of CXCR1 and CXCR2 are distinct: the N-loop of CXCL8 is the major determinant for CXCR1 activation, whereas the N-terminus of CXCL8 (ELR and CXC) is essential for CXCR2 activation. We also found that activation of CXCR1 cross-desensitized CXCR2 responses in human neutrophils co-expressing both receptors, indicating that these novel CXCR1 agonists represent a new class of anti-inflammatory agents. Further, these selective CXCR1 agonists will aid at elucidating the functional significance of CXCR1 in vivo under pathophysiological conditions

In Silico Elucidation of the Recognition Dynamics of Ubiquitin

Elucidation of the mechanism of biomacromolecular recognition events has been a topic of intense interest over the past century. The inherent dynamic nature of both protein and ligand molecules along with the continuous reshaping of the energy landscape during the binding process renders it difficult to characterize this process at atomic detail. Here, we investigate the recognition dynamics of ubiquitin via microsecond all-atom molecular dynamics simulation providing both thermodynamic and kinetic information. The high-level of consistency found with respect to experimental NMR data lends support to the accuracy of the in silico representation of the conformational substates and their interconversions of free ubiquitin. Using an energy-based reweighting approach, the statistical distribution of conformational states of ubiquitin is monitored as a function of the distance between ubiquitin and its binding partner Hrs-UIM. It is found that extensive and dense sampling of conformational space afforded by the µs MD trajectory is essential for the elucidation of the binding mechanism as is Boltzmann sampling, overcoming inherent limitations of sparsely sampled empirical ensembles. The results reveal a population redistribution mechanism that takes effect when the ligand is at intermediate range of 1–2 nm from ubiquitin. This mechanism, which may be depicted as a superposition of the conformational selection and induced fit mechanisms, also applies to other binding partners of ubiquitin, such as the GGA3 GAT domain