Conformational Dynamics and Structural Plasticity Play Critical Roles in the Ubiquitin Recognition of a UIM Domain (original) (raw)

Weak Long-Range Correlated Motions in a Surface Patch of Ubiquitin Involved in Molecular Recognition

Journal of the American Chemical Society, 2011

Long-range correlated motions in proteins are candidate mechanisms for processes that require information transfer across protein structures, such as allostery and signal transduction. However, the observation of backbone correlations between distant residues has remained elusive, and only local correlations have been revealed using residual dipolar couplings measured by NMR spectroscopy. In this work, we experimentally identified and characterized collective motions spanning four β-strands separated by up to 15 Å in ubiquitin. The observed correlations link molecular recognition sites and result from concerted conformational changes that are in part mediated by the hydrogenbonding network.

Ubiquitin chain conformation regulates recognition and activity of interacting proteins

Mechanisms of protein recognition have been extensively studied for single-domain proteins 1 , but are less well characterized for dynamic multidomain systems. Ubiquitin chains represent a biologically important multidomain system that requires recognition by structurally diverse ubiquitin-interacting proteins 2,3 . Ubiquitin chain conformations in isolation are often different from conformations observed in ubiquitin-interacting protein complexes, indicating either great dynamic flexibility or extensive chain remodelling upon binding. Using single-molecule fluorescence resonance energy transfer, we show that Lys 63-, Lys 48-and Met 1-linked diubiquitin exist in several distinct conformational states in solution. Lys 63-and Met 1-linked diubiquitin adopt extended 'open' and more compact 'closed' conformations, and ubiquitin-binding domains and deubiquitinases (DUBs) select pre-existing conformations. By contrast, Lys 48-linked diubiquitin adopts predominantly compact conformations. DUBs directly recognize existing conformations, but may also remodel ubiquitin chains to hydrolyse the isopeptide bond. Disruption of the Lys 48-diubiquitin interface changes conformational dynamics and affects DUB activity. Hence, conformational equilibria in ubiquitin chains provide an additional layer of regulation in the ubiquitin system, and distinct conformations observed in differently linked polyubiquitin may contribute to the specificity of ubiquitin-interacting proteins.

Ubiquitin Interacting Motifs: Duality Between Structured and Disordered Motifs

Frontiers in Molecular Biosciences, 2021

Ubiquitin is a small protein at the heart of many cellular processes, and several different protein domains are known to recognize and bind ubiquitin. A common motif for interaction with ubiquitin is the Ubiquitin Interacting Motif (UIM), characterized by a conserved sequence signature and often found in multi-domain proteins. Multi-domain proteins with intrinsically disordered regions mediate interactions with multiple partners, orchestrating diverse pathways. Short linear motifs for binding are often embedded in these disordered regions and play crucial roles in modulating protein function. In this work, we investigated the structural propensities of UIMs using molecular dynamics simulations and NMR chemical shifts. Despite the structural portrait depicted by X-crystallography of stable helical structures, we show that UIMs feature both helical and intrinsically disordered conformations. Our results shed light on a new class of disordered UIMs. This group is here exemplified by th...

Slow conformational exchange and overall rocking motion in ubiquitin protein crystals

2017

Proteins perform their functions in solution but their structures are most frequently studied inside crystals. Here we probe how the crystal packing alters microsecond dynamics, using solid-state NMR measurements and multi-microsecond MD simulations of different crystal forms of ubiquitin. In particular, NEar-Rotary-resonance Relaxation Dispersion (NERRD) experiments probe angular backbone motion, while Bloch-McConnell Relaxation Dispersion data report on fluctuations of the local electronic environment. These experiments and simulations reveal that the packing of the protein can significantly alter the thermodynamics and kinetics of local conformational exchange. Moreover, we report small-amplitude reorientational motion of protein molecules in the crystal lattice with a ~3-5 degrees amplitude on a tens-of-microseconds time scale in one of the crystals, but not in others. An intriguing possibility arises that overall motion is to some extent coupled to local dynamics. Our study hig...

The mechanical stability of ubiquitin is linkage dependent

Nature Structural Biology, 2003

Naturally occurring ubiquitin chains are found to be either N-C-linked or linked between their C terminus and an exposed lysine residue that can be Lys63, Lys48, Lys29 or Lys11, wherein each ubiquitin chain is assembled with a single type of linkage between the ubiquitin monomers 1-4 . These ubiquitin chains offer a new opportunity for studying the mechanical stability of a protein under conditions in which the mechanical force is applied to the protein through different linkages. Our studies made use of single-molecule force spectroscopy 5,6 and SMD techniques 7 to examine how the type of linkage affects the mechanical stability of native ubiquitin chains. Singlemolecule techniques have been used to examine the mechanical stability of native proteins such as titin 5 and fibronectin 6 . However, the heterogeneous mixture of modules found in these proteins made it difficult to assign mechanical properties to individual modules. The engineering of proteins made of tandem repeats of an identical module, called polyproteins, has permitted a module-by-module dissection of the mechanical properties of native proteins 8-15 . However, polyproteins are rare in nature, with the exception of ubiquitin. The different polyubiquitin linkages have important functions in cell signaling and target proteins to different pathways. For example, protein substrates tethered by a Lys48-linked polyubiquitin are specifically tagged for degradation by the proteasome 16 . Here we use single-molecule atomic force microscopy (AFM) techniques 10,11 to study the effect of a mechanical force on polyubiquitin chains. We study the mechanical properties of two types of ubiquitin polyproteins, an N-C-linked polyprotein composed of nine repeats (N-Ub 9 ; ) and a Lys48-C-linked polyprotein ranging from two to seven repeats (Lys48-Ub 2-7 ).

The role of binding site on the mechanical unfolding mechanism of ubiquitin

Scientific reports, 2015

We apply novel atomistic simulations based on potential energy surface exploration to investigate the constant force-induced unfolding of ubiquitin. At the experimentally-studied force clamping level of 100 pN, we find a new unfolding mechanism starting with the detachment between β5 and β3 involving the binding site of ubiquitin, the Ile44 residue. This new unfolding pathway leads to the discovery of new intermediate configurations, which correspond to the end-to-end extensions previously seen experimentally. More importantly, it demonstrates the novel finding that the binding site of ubiquitin can be responsible not only for its biological functions, but also its unfolding dynamics. We also report in contrast to previous single molecule constant force experiments that when the clamping force becomes smaller than about 300 pN, the number of intermediate configurations increases dramatically, where almost all unfolding events at 100 pN involve an intermediate configuration. By direc...

Residual dipolar couplings as a tool to study molecular recognition of ubiquitin

Biochemical Society Transactions, 2008

RDCs (residual dipolar couplings) in NMR spectroscopy provide information about protein dynamics complementary to NMR relaxation methods, especially in the previously inaccessible time window between the protein correlation time τc and 50 μs. For ubiquitin, new modes of motion of the protein backbone could be detected using RDC-based techniques. An ensemble of ubiquitin based on these RDC values is found to comprise all different conformations that ubiquitin adopts upon binding to different recognition proteins. These conformations in protein–protein complexes had been derived from 46 X-ray structures. Thus, for ubiquitin recognition by other proteins, conformational selection rather than induced fit seems to be the dominant mechanism.

Dissecting the mechanical unfolding of ubiquitin

Proceedings of the National Academy of Sciences, 2005

The unfolding behavior of ubiquitin under the influence of a stretching force recently was investigated experimentally by single-molecule constant-force methods. Many observed unfolding traces had a simple two-state character, whereas others showed clear evidence of intermediate states. Here, we use Monte Carlo simulations to investigate the force-induced unfolding of ubiquitin at the atomic level. In agreement with experimental data, we find that the unfolding process can occur either in a single step or through intermediate states. In addition to this randomness, we find that many quantities, such as the frequency of occurrence of intermediates, show a clear systematic dependence on the strength of the applied force. Despite this diversity, one common feature can be identified in the simulated unfolding events, which is the order in which the secondary-structure elements break. This order is the same in two-and three-state events and at the different forces studied. The observed order remains to be verified experimentally but appears physically reasonable. all-atom model ͉ force-induced unfolding ͉ Monte Carlo simulation T he 76-residue protein ubiquitin fulfills many important regulatory functions in eukaryotic cells through its covalent attachment to other proteins (1, 2). In many cases, the ubiquitin tag consists of a chain of ubiquitin domains (polyubiquitin), which is formed by linkages between an exposed lysine side chain of the last ubiquitin of a growing chain and the C terminus of a new ubiquitin. The fate of a polyubiquitin-tagged protein depends on the linkage. For example, Lys-48-C-linked polyubiquitin marks the protein substrate for proteasomal degradation (3).