A variety of membraneless organelles, often termed “biological condensates”, play an important role in the regulation of cellular processes such as gene transcription, translation, and protein quality control. On the basis of experimental and theoretical investigations, liquid–liquid phase separation (LLPS) has been proposed as a possible mechanism for the origin of biological condensates. LLPS requires multivalent macromolecules that template the formation of long-range, intermolecular interaction networks and results in the formation of condensates with defined composition and material properties. Multivalent interactions driving LLPS exhibit a wide range of modes from highly stereospecific to nonspecific and involve both folded and disordered regions. Multidomain proteins serve as suitable macromolecules for promoting phase separation and achieving disparate functions due to their potential for multivalent interactions and regulation. Here, we aim to highlight the influence of the domain architecture and interdomain interactions on the phase separation of multidomain protein condensates. First, the general principles underlying these interactions are illustrated on the basis of examples of multidomain proteins that are predominantly associated with nucleic acid binding and protein quality control and contain both folded and disordered regions. Next, the examples showcase how LLPS properties of folded and disordered regions can be leveraged to engineer multidomain constructs that form condensates with the desired assembly and functional properties. Finally, we highlight the need for improvements in coarse-grained computational models that can provide molecular-level insights into multidomain protein condensates in conjunction with experimental efforts.

The deamidase OspI from enteric bacteria Shigella flexneri deamidates a glutamine residue in the host ubiquitin-conjugating enzyme UBC13 and converts it to glutamate (Q100E). Consequently, its polyubiquitination activity in complex with the RING-finger ubiquitin ligase TRAF6 and the downstream NF-κB inflammatory response is silenced. The precise role of deamidation in silencing the UBC13/TRAF6 complex is unknown. We report that deamidation inhibits the interaction between UBC13 and TRAF6 RING-domain (TRAF6RING) by perturbing both the native and transient interactions. Deamidation creates a new intramolecular salt-bridge in UBC13 that competes with a critical intermolecular salt-bridge at the native UBC13/TRAF6RING interface. Moreover, the salt-bridge competition prevents transient interactions necessary to form a typical UBC13/RING complex. Repulsion between E100 and the negatively charged surface of RING also prevents transient interactions in the UBC13/RING complex. Our findings highlight a mechanism wherein a post-translational modification perturbs the conformation and stability of transient complexes to inhibit protein-protein association.

TAR DNA-binding protein 43 (TDP-43) is involved in key processes in RNA metabolism and is frequently implicated in many neurodegenerative diseases, including amyotrophic lateral sclerosis and frontotemporal dementia. The prion-like, disordered C-terminal domain (CTD) of TDP-43 is aggregation-prone, can undergo liquid-liquid phase separation (LLPS) in isolation, and is critical for phase separation (PS) of the full-length protein under physiological conditions. While a short conserved helical region (CR, spanning residues 319-341) promotes oligomerization and is essential for LLPS, aromatic residues in the flanking disordered regions (QN-rich, IDR1/2) are also found to play a critical role in PS and aggregation. Compared with other phase-separating proteins, TDP-43 CTD has a notably distinct sequence composition including many aliphatic residues such as methionine and leucine. Aliphatic residues were previously suggested to modulate the apparent viscosity of the resulting phases, but their direct contribution toward CTD phase separation has been relatively ignored. Using multiscale simulations coupled with in vitro saturation concentration (c sat ) measurements, we identified the importance of aromatic residues while also suggesting an essential role for aliphatic methionine residues in promoting single-chain compaction and LLPS. Surprisingly, NMR experiments showed that transient interactions involving phenylalanine and methionine residues in the disordered flanking regions can directly enhance site-specific, CR-mediated intermolecular association. Overall, our work highlights an underappreciated mode of biomolecular recognition, wherein both transient and site-specific hydrophobic interactions act synergistically to drive the oligomerization and phase separation of a disordered, low-complexity domain.

26The deamidase OspI from enteric bacteria Shigella flexneri deamidates a glutamine residue in the 27 host ubiquitin-conjugating enzyme UBC13 and converts it to glutamate (Q100E). Consequently, its 28 polyubiquitination activity in complex with the RING-finger ubiquitin ligase TRAF6 and the downstream 29 NF-B inflammatory response is inactivated. The precise role of deamidation in inactivating the 30 UBC13/TRAF6 complex is unknown. We report that deamidation inhibits the interaction between 31 UBC13 and TRAF6 RING-domain (TRAF6 RING ) by perturbing both the native and transient 32 interactions. Deamidation creates a new intramolecular salt-bridge in UBC13 that competes with a 33 critical intermolecular salt-bridge at the native UBC13/TRAF6 RING interface. Moreover, the salt-bridge 34 competition prevents transient interactions necessary to form a typical UBC13/RING complex. 35Repulsion between E100 and the negatively charged surface of RING also prevents transient 36 interactions in the UBC13/RING complex. Our findings highlight a mechanism where a post-37 translational modification perturbs the conformation and stability of transient complexes to inhibit 38 protein-protein association. 39 40 41 42 43 44 45finger E3s like TRAF6 to synthesize K63-linked poly-Ub chains that function to activate DNA repair or 72 immune response (Matsuzawa et al. 2007). Apart from the E3s, UBC13 also binds a co-factor MMS2, 73 which does not activate UBC13 but maintains the linkage specificity of the poly-Ub chains synthesized 74 by UBC13 (Branigan et al. 2015). 75In this study, we have investigated the mechanisms underlying the inactivation of UBC13 76 upon deamidation using NMR spectroscopy, molecular dynamics (MD) simulations, and in-vitro 77 ubiquitination assays. We report that deamidation weakens the non-covalent interaction of UBC13 78 with RING-finger domain of TRAF6 (TRAF6 RING ), without perturbing UBC13 structure or the enzymatic 79 activity of UBC13. However, the underlying cause of reduced interaction is nonintuitive since Q100 is 80 in the vicinity of UBC13/TRAF6 RING interface but does not form any contact with the TRAF6 RING . 81Further studies showed that deamidation disrupts the interaction between UBC13 and TRAF6 RING by 82 three mechanisms: i) A new intramolecular R14/E100 salt-bridge appears in dUBC13, which competes 83 with a critical intermolecular salt-bridge in the native complex, ii) the salt-bridge competition also 84 perturbs the UBC13/TRAF6 RING transient complexes to inhibit association, and iii) repulsion between 85 the negatively charged E100 and the negatively charged interface of TRAF6 RING perturbs the transient 86 complexes to reduce association between UBC13 and TRAF6 RING . The effect of each mechanism on 87 the binding was confirmed by binding studies using appropriate substitutions in either UBC13 or 88 TRAF6 RING . The impact of deamidation on transient interactions was also observed using another 89 RING domain from RNF38, indicating that the mechanism could be ubiquitous for UBC13/RING 90 c...