<i>Mycobacterium tuberculosis</i>
 copper-regulated protein SocB is an intrinsically disordered protein that folds upon interaction with a synthetic phospholipid bilayer

Nowicka, Urszula; Hoffman, Morgan; Randles, Leah; Shi, Xiaoshan; Khavrutskii, Lyuba; Stefanisko, Karen; Tarasova, Nadya I.; Darwin, K. Heran; Walters, Kylie J.

doi:10.1002/prot.24970

Cited by 4 publications

(5 citation statements)

References 27 publications

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“…Diubiquitin with each of the eight ubiquitin linkage types (M1, K6, K11, K27, K29, K33, K48 and K63) was incubated with Ni-NTA resin containing pre-bound His-tagged hRpn10 protein and interaction detected by immunoblotting with anti-ubiquitin antibody following the removal of unbound protein (Figure 1a). Intrinsically disordered protein SocB with a His-tag [80] was used as a negative control with K48 diubiquitin. Indeed, hRpn10 interacted with all ubiquitin chain types, with strongest affinity for K48 linked diubiquitin (Figure 1a), the preferred chain type also for Rpn1 and Rpn13 [6,45].…”

Section: Results Hrpn10 Prefers K48 and K11 Linked Ubiquitinsmentioning

confidence: 99%

“…The resin was then incubated with 500 pmol of M1, K6, K11, K27, K29, K33, K48, and K63 diubiquitin for one hour. Intrinsically disordered protein SocB with a C-terminal His-tag [80] was used as a negative control with K48 diubiquitin. Unbound protein was removed by extensive washing in the above buffer.…”

Section: His Pull-down Assaysmentioning

confidence: 99%

“…Immunoblotting was done with antibody against ubiquitin (anti-ubiquitin) (top) or polyhistidine (anti-His) (middle). Intrinsically disordered protein SocB-His [80] was used as a negative control with K48 diubiquitin, as indicated. Direct loading for 15% of the diubiquitin input for each chain type with immunoblotting by ubiquitin-specific antibody is included (bottom).…”

Section: Model Structure Of Uim-1 Complexed With S65-phosphorylated Ubiquitin or Parkin Ublmentioning

confidence: 99%

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Structure of hRpn10 Bound to UBQLN2 UBL Illustrates Basis for Complementarity between Shuttle Factors and Substrates at the Proteasome

Chen

Ebelle

Wright

et al. 2019

Journal of Molecular Biology

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The 26S proteasome is a highly complex 2.5 MDa molecular machine responsible for regulated protein degradation. Proteasome substrates are typically marked by ubiquitination for recognition at receptor sites contributed by Rpn1/S2/PSMD2, Rpn10/S5a, and Rpn13/Adrm1. Each receptor site can bind substrates directly by engaging conjugated ubiquitin chains or indirectly by binding to shuttle factors Rad23/HR23, Dsk2/PLIC/UBQLN, or Ddi1, which contain a ubiquitin-like domain (UBL) that adopts the ubiquitin fold. Previous structural studies have defined how each of the proteasome receptor sites bind to ubiquitin chains as well as some of the interactions that occur with the shuttle factors. Here, we define how hRpn10 binds to the UBQLN2 UBL domain, solving the structure of this complex by NMR, and determine affinities for each UIM region by a titration experiment. UBQLN2 UBL exhibits 25-fold stronger affinity for the N-terminal UIM-1 over UIM-2 of hRpn10. Moreover, we discover that UBQLN2 UBL is fine-tuned for the hRpn10 UIM-1 site over the UIM-2 site by taking advantage of the additional contacts made available through the longer UIM-1 helix. We also test hRpn10 versatility for the various ubiquitin chains to find less specificity for any particular linkage type compared to hRpn1 and hRpn13, as expected from the flexible linker region that connects the two UIMs; nonetheless, hRpn10 does exhibit some preference for K48 and K11 linkages. Altogether, these results provide new insights into the highly complex and complementary roles of the proteasome receptor sites and shuttle factors.

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Section: Results Hrpn10 Prefers K48 and K11 Linked Ubiquitinsmentioning

confidence: 99%

Section: His Pull-down Assaysmentioning

confidence: 99%

Section: Model Structure Of Uim-1 Complexed With S65-phosphorylated Ubiquitin or Parkin Ublmentioning

confidence: 99%

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Structure of hRpn10 Bound to UBQLN2 UBL Illustrates Basis for Complementarity between Shuttle Factors and Substrates at the Proteasome

Chen

Ebelle

Wright

et al. 2019

Journal of Molecular Biology

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“…The resin was next incubated with 800 pmol of M1-, K6-, K11-, K27-, K29-, K33-, K48-, and K63-linked diubiquitin for one hour. Intrinsically disordered protein SocB with a C-terminal His-tag (49) was used as a negative control with K48-linked diubiquitin. Unbound protein was removed by extensive washing in the above buffer.…”

Section: Methodsmentioning

confidence: 99%

“…Immunoblotting was done with anti-ubiquitin (top, left) or anti-His (middle, left) antibodies. Intrinsically disordered protein SocB-His (49) was used as a negative control with K48 diubiquitin, as indicated. Direct loading for 15% of the diubiquitin input for each chain type with immunoblotting by anti-ubiquitin antibody is included (bottom, left).…”

Section: Figurementioning

confidence: 99%

Rpn1 provides adjacent receptor sites for substrate binding and deubiquitination by the proteasome

Shi

Chen

Elsasser

et al. 2016

Science

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260

355

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Structured Abstract INTRODUCTION The ubiquitin-proteasome system comprises hundreds of distinct pathways of degradation, which converge at the step of ubiquitin recognition by the proteasome. Five proteasomal ubiquitin receptors have been identified, two that are intrinsic to the proteasome (Rpn10 and Rpn13) and three reversibly associated proteasomal ubiquitin receptors (Rad23, Dsk2, and Ddi1). RATIONALE We found that the five known proteasomal ubiquitin receptors of yeast are collectively nonessential for ubiquitin recognition by the proteasome. We therefore screened for additional ubiquitin receptors in the proteasome and identified subunit Rpn1 as a candidate. We used nuclear magnetic resonance (NMR) spectroscopy to characterize the structure of the binding site within Rpn1, which we term the T1 site. Mutational analysis of this site showed its functional importance within the context of intact proteasomes. T1 binds both ubiquitin and ubiquitin-like (UBL) proteins, in particular the substrate-delivering shuttle factor Rad23. A second site within the Rpn1 toroid, T2, recognizes the UBL domain of deubiquitinating enzyme Ubp6, as determined by hydrogen-deuterium exchange mass spectrometry analysis and validated by amino acid substitution and functional assays. The Rpn1 toroid thus serves a critical scaffolding role within the proteasome, helping to assemble multiple proteasome cofactors as well as substrates. RESULTS Our results indicate that proteasome subunit Rpn1 can recognize both ubiquitin and UBL domains of substrate shuttling factors that themselves bind ubiquitin and function as reversibly-associated proteasomal ubiquitin receptors. Recognition is mediated by the T1 site within the Rpn1 toroid, which supports proteasome function in vivo. We found that the capacity of T1 to recognize both ubiquitin and UBL proteins was shared with Rpn10 and Rpn13. The surprising multiplicity of ubiquitin-recognition domains within the proteasome may promote enhanced, multipoint binding of ubiquitin chains. The structures of the T1 site in its free state and complexed with monoubiquitin or K48-linked diubiquitin were solved, revealing that three neighboring outer helices from the T1 toroid engage two ubiquitins. This binding mode leads to a preference for certain ubiquitin chain types, especially K6- and K48-linked chains, in a distinct configuration that can position substrates close to the entry port of the proteasome. The fate of proteasome-docked ubiquitin conjugates is determined by a competition between deubiquitination and substrate degradation. We find that proximal to the T1 site within the Rpn1 toroid is a second UBL-binding site, T2, that does not assist in ubiquitin chain recognition, but rather in chain disassembly, by binding to the UBL domain of deubiquitinating enzyme Ubp6. Importantly, the UBL interactors at T1 and T2 are distinct, assigning substrate localization to T1 and substrate deubiquitination to T2. CONCLUSION A ligand-binding hotspot was identified in the Rpn1 toroid, consisting of two adj...

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Structural and Biophysical properties of therapeutically important proteins Rv1509 and Rv2231A of Mycobacterium tuberculosis

Rastogi

Zarin

Alam

et al. 2023

International Journal of Biological Macromolecules

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Mycobacterium tuberculosis copper-regulated protein SocB is an intrinsically disordered protein that folds upon interaction with a synthetic phospholipid bilayer

Cited by 4 publications

References 27 publications

Structure of hRpn10 Bound to UBQLN2 UBL Illustrates Basis for Complementarity between Shuttle Factors and Substrates at the Proteasome

Structure of hRpn10 Bound to UBQLN2 UBL Illustrates Basis for Complementarity between Shuttle Factors and Substrates at the Proteasome

Rpn1 provides adjacent receptor sites for substrate binding and deubiquitination by the proteasome

Structural and Biophysical properties of therapeutically important proteins Rv1509 and Rv2231A of Mycobacterium tuberculosis

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