Saturation scanning of ubiquitin variants reveals a common hot spot for binding to USP2 and USP21.


Abstract

A detailed understanding of the molecular mechanisms whereby ubiquitin (Ub) recognizes enzymes in the Ub proteasome system is crucial for understanding the biological function of Ub. Many structures of Ub complexes have been solved and, in most cases, reveal a large structural epitope on a common face of the Ub molecule. However, owing to the generally weak nature of these interactions, it has been difficult to map in detail the functional contributions of individual Ub side chains to affinity and specificity. Here we took advantage of Ub variants (Ubvs) that bind tightly to particular Ub-specific proteases (USPs) and used phage display and saturation scanning mutagenesis to comprehensively map functional epitopes within the structural epitopes. We found that Ubvs that bind to USP2 or USP21 contain a remarkably similar core functional epitope, or "hot spot," consisting mainly of positions that are conserved as the wild type sequence, but also some positions that prefer mutant sequences. The Ubv core functional epitope contacts residues that are conserved in the human USP family, and thus it is likely important for the interactions of Ub across many family members.

Submission Details

ID: VkahB8tt

Submitter: Marie Ary

Submission Date: June 14, 2017, 5:50 p.m.

Version: 1

Publication Details
Leung I;Dekel A;Shifman JM;Sidhu SS,Proc Natl Acad Sci U S A (2016) Saturation scanning of ubiquitin variants reveals a common hot spot for binding to USP2 and USP21. PMID:27436899
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
5L9U 2016-06-11T00:00:00+0000 6.4 Model of human Anaphase-promoting complex/Cyclosome (APC/C-CDH1) with a cross linked Ubiquitin variant-substrate-UBE2C (UBCH10) complex representing key features of multiubiquitination
6ZVK 2020-07-24T00:00:00+0000 3.49 The Halastavi arva virus (HalV) intergenic region IRES promotes translation by the simplest possible initiation mechanism
1BT0 1998-09-02T00:00:00+0000 1.7 STRUCTURE OF UBIQUITIN-LIKE PROTEIN, RUB1
4V8M 2012-12-09T00:00:00+0000 5.57 High-resolution cryo-electron microscopy structure of the Trypanosoma brucei ribosome
4V6W 2013-02-27T00:00:00+0000 6.0 Structure of the D. melanogaster 80S ribosome
4V6W 2013-02-27T00:00:00+0000 6.0 Structure of the D. melanogaster 80S ribosome
5OHM 2017-07-17T00:00:00+0000 3.8 K33-specific affimer bound to K33 diUb
4ADX 2012-01-04T00:00:00+0000 6.6 The Cryo-EM Structure of the Archaeal 50S Ribosomal Subunit in Complex with Initiation Factor 6
4V8P 2011-09-14T00:00:00+0000 3.52 T.thermophila 60S ribosomal subunit in complex with initiation factor 6.
6NYA 2019-02-11T00:00:00+0000 2.06 Crystal Structure of ubiquitin E1 (Uba1) in complex with Ubc3 (Cdc34) and ubiquitin

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
97.4 Ubiquitin variant Ubv.2.1 P31753 RS27A_ASPOF
100.0 Ubiquitin variant Ubv.2.1 P84589 UBIQ_LUMTE
91.4 Ubiquitin variant Ubv.2.1 P59272 RS27A_DAUCA
91.7 Ubiquitin variant Ubv.2.1 P0C8R3 RS27B_SCHPO
91.7 Ubiquitin variant Ubv.2.1 P0C016 RS27A_SCHPO
91.7 Ubiquitin variant Ubv.2.1 P27923 RS27A_MAIZE
91.7 Ubiquitin variant Ubv.2.1 P47905 RS27A_LUPAL
91.7 Ubiquitin variant Ubv.2.1 P0CG87 RS272_HORVU
91.7 Ubiquitin variant Ubv.2.1 P0CG86 RS271_HORVU
91.7 Ubiquitin variant Ubv.2.1 P59233 R27AC_ARATH
91.7 Ubiquitin variant Ubv.2.1 P51431 R27AB_ORYSJ
91.7 Ubiquitin variant Ubv.2.1 P59232 R27AB_ARATH
91.7 Ubiquitin variant Ubv.2.1 Q9ARZ9 R27AA_ORYSJ
90.8 Ubiquitin variant Ubv.21.4 P21899 RL40_TRYBB
90.8 Ubiquitin variant Ubv.21.4 Q3E7K8 UBQ12_ARATH
92.1 Ubiquitin variant Ubv.21.4 P0CG70 UBI4P_NEUCR
92.0 Ubiquitin variant Ubv.21.4 P14799 RS27A_NEUCR
92.1 Ubiquitin variant Ubv.21.4 P14795 RL40_TRYCR
92.1 Ubiquitin variant Ubv.21.4 P0C224 RL40_NEUCR
92.1 Ubiquitin variant Ubv.21.4 P49633 RL40_ACACA
92.1 Ubiquitin variant Ubv.21.4 P0CH27 RL402_TRYCR
90.8 Ubiquitin variant Ubv.21.4 P22589 UBIQP_PHYIN
90.8 Ubiquitin variant Ubv.21.4 P42739 UBIQP_ACEPE
90.8 Ubiquitin variant Ubv.21.4 P0C032 RUB3_ORYSJ
91.9 Ubiquitin variant Ubv.2.1 P14797 RS27A_DICDI
92.1 Ubiquitin variant Ubv.21.4 P0CH32 UBQ4_ARATH
92.1 Ubiquitin variant Ubv.21.4 Q58G87 UBQ3_ORYSJ
92.1 Ubiquitin variant Ubv.21.4 Q1EC66 UBQ3_ARATH
92.1 Ubiquitin variant Ubv.21.4 Q3E7T8 UBQ14_ARATH
92.1 Ubiquitin variant Ubv.21.4 P0CH33 UBQ11_ARATH
92.1 Ubiquitin variant Ubv.21.4 Q8H159 UBQ10_ARATH
92.1 Ubiquitin variant Ubv.21.4 P69326 UBIQ_WHEAT
92.1 Ubiquitin variant Ubv.21.4 P69317 UBIQ_LUPPO
92.1 Ubiquitin variant Ubv.21.4 P69313 UBIQ_HELAN
92.1 Ubiquitin variant Ubv.21.4 P69310 UBIQ_AVESA
92.1 Ubiquitin variant Ubv.21.4 P69325 UBIQP_SOYBN
92.1 Ubiquitin variant Ubv.21.4 P69322 UBIQP_PEA
92.1 Ubiquitin variant Ubv.21.4 P69315 UBIQP_LINUS
92.1 Ubiquitin variant Ubv.21.4 P0CG83 UBIQP_HORVU
92.1 Ubiquitin variant Ubv.21.4 P69309 UBIQP_AVEFA
93.4 Ubiquitin variant Ubv.21.4 P0CG88 UBIQJ_DICDI
93.4 Ubiquitin variant Ubv.21.4 P0CG80 UBIQI_DICDI
93.4 Ubiquitin variant Ubv.21.4 P0CG81 UBIQH_DICDI
93.4 Ubiquitin variant Ubv.21.4 P0CG79 UBIQG_DICDI
93.4 Ubiquitin variant Ubv.21.4 P0CG78 UBIQF_DICDI
93.4 Ubiquitin variant Ubv.21.4 P0CG77 UBIQD_DICDI
93.4 Ubiquitin variant Ubv.21.4 P0CG76 UBIQA_DICDI
92.1 Ubiquitin variant Ubv.21.4 P0CG63 UBI4P_YEAST
92.1 Ubiquitin variant Ubv.21.4 P0CG72 UBI4P_SCHPO
92.1 Ubiquitin variant Ubv.21.4 P0CG84 UBI4P_NICSY
92.1 Ubiquitin variant Ubv.21.4 P0CG75 UBI4P_KLULA
92.1 Ubiquitin variant Ubv.21.4 P0CG74 UBI4P_CANAX
92.1 Ubiquitin variant Ubv.21.4 P0CH05 UBI2P_PETCR
92.1 Ubiquitin variant Ubv.21.4 P0CH04 UBI1P_PETCR
92.1 Ubiquitin variant Ubv.21.4 P0CG85 UBI1P_NICSY
92.1 Ubiquitin variant Ubv.21.4 P0CG73 UBI1P_CANAX
92.1 Ubiquitin variant Ubv.21.4 P0C031 RUB2_ORYSJ
92.1 Ubiquitin variant Ubv.21.4 Q8RUC6 RUB2_ARATH
92.1 Ubiquitin variant Ubv.21.4 P0C030 RUB1_ORYSJ
92.1 Ubiquitin variant Ubv.21.4 P0C073 RUB1_DESAN
92.1 Ubiquitin variant Ubv.21.4 Q9SHE7 RUB1_ARATH
92.0 Ubiquitin variant Ubv.21.4 P05759 RS31_YEAST
92.1 Ubiquitin variant Ubv.21.4 P62981 RS27A_SOLTU
92.1 Ubiquitin variant Ubv.21.4 P62980 RS27A_SOLLC
92.0 Ubiquitin variant Ubv.21.4 P69061 RS27A_KLULA
92.1 Ubiquitin variant Ubv.21.4 P49636 RL40_NICSY
93.4 Ubiquitin variant Ubv.21.4 P14794 RL40_DICDI
92.1 Ubiquitin variant Ubv.21.4 P40909 RL40_CRYNJ
92.1 Ubiquitin variant Ubv.21.4 P51423 RL40_BRARP
92.1 Ubiquitin variant Ubv.21.4 P0CH09 RL40B_YEAST
92.1 Ubiquitin variant Ubv.21.4 P0CH35 RL40B_ORYSJ
92.1 Ubiquitin variant Ubv.21.4 Q42202 RL40B_ARATH
92.1 Ubiquitin variant Ubv.21.4 P0CH08 RL40A_YEAST
92.1 Ubiquitin variant Ubv.21.4 P0CH34 RL40A_ORYSJ
92.1 Ubiquitin variant Ubv.21.4 B9DHA6 RL40A_ARATH
92.1 Ubiquitin variant Ubv.21.4 P0CH07 RL402_SCHPO
92.1 Ubiquitin variant Ubv.21.4 P0CH06 RL401_SCHPO
92.1 Ubiquitin variant Ubv.21.4 P59271 R27AA_ARATH
93.4 Ubiquitin variant Ubv.21.4 P42740 UBIQP_AGLNE
93.4 Ubiquitin variant Ubv.21.4 P19848 UBIQ_COPCO
93.4 Ubiquitin variant Ubv.21.4 P0CG82 UBIQP_TETPY
93.4 Ubiquitin variant Ubv.21.4 P0DJ25 RL40_TETTS
93.4 Ubiquitin variant Ubv.21.4 P33190 RL40_TETPY
93.4 Ubiquitin variant Ubv.21.4 P0CH10 RL403_CHLRE
93.4 Ubiquitin variant Ubv.21.4 P0CH11 RL401_CHLRE
93.2 Ubiquitin variant Ubv.2.1 P69200 RL40_LEITA
93.4 Ubiquitin variant Ubv.21.4 P69201 RL40_LEIMA
94.7 Ubiquitin variant Ubv.21.4 P59669 UBIQP_GEOCY
94.7 Ubiquitin variant Ubv.21.4 P0CG71 UBIQ1_CAEEL
94.7 Ubiquitin variant Ubv.21.4 P49632 RL40_CAEEL
94.7 Ubiquitin variant Ubv.21.4 Q8SWD4 UBIQ_ENCCU
94.7 Ubiquitin variant Ubv.21.4 P23398 UBIQP_STRPU
94.7 Ubiquitin variant Ubv.21.4 P46575 RL40_EIMBO
96.1 Ubiquitin variant Ubv.21.4 P62975 UBIQ_RABIT
96.1 Ubiquitin variant Ubv.21.4 P68197 UBIQ_CERCA
96.1 Ubiquitin variant Ubv.21.4 Q865C5 UBIQ_CAMDR
96.1 Ubiquitin variant Ubv.21.4 P62972 UBIQP_XENLA
96.1 Ubiquitin variant Ubv.21.4 P0CG69 UBIQP_DROME
96.1 Ubiquitin variant Ubv.21.4 P62976 UBIQP_CRIGR
96.1 Ubiquitin variant Ubv.21.4 Q63429 UBC_RAT
96.1 Ubiquitin variant Ubv.21.4 P0CG61 UBC_PONPY
96.1 Ubiquitin variant Ubv.21.4 P0CG68 UBC_PIG
96.1 Ubiquitin variant Ubv.21.4 P0CG64 UBC_PANTR
96.1 Ubiquitin variant Ubv.21.4 P0CG50 UBC_MOUSE
96.1 Ubiquitin variant Ubv.21.4 P0CG48 UBC_HUMAN
96.1 Ubiquitin variant Ubv.21.4 P0CG66 UBC_GORGO
96.1 Ubiquitin variant Ubv.21.4 P0CH28 UBC_BOVIN
96.1 Ubiquitin variant Ubv.21.4 P0CG55 UBB_SHEEP
96.1 Ubiquitin variant Ubv.21.4 P0CG51 UBB_RAT
96.1 Ubiquitin variant Ubv.21.4 P0CG60 UBB_PONPY
96.1 Ubiquitin variant Ubv.21.4 P0CG65 UBB_PANTR
96.1 Ubiquitin variant Ubv.21.4 P0CG49 UBB_MOUSE
96.1 Ubiquitin variant Ubv.21.4 P0CG47 UBB_HUMAN
96.1 Ubiquitin variant Ubv.21.4 Q8MKD1 UBB_HORSE
96.1 Ubiquitin variant Ubv.21.4 P0CG67 UBB_GORGO
96.1 Ubiquitin variant Ubv.21.4 P0CG62 UBB_CHICK
96.1 Ubiquitin variant Ubv.21.4 P0CG54 UBB_CAVPO
96.1 Ubiquitin variant Ubv.21.4 P0CG53 UBB_BOVIN
96.1 Ubiquitin variant Ubv.21.4 P68203 RS27A_SPOFR
96.0 Ubiquitin variant Ubv.21.4 P62982 RS27A_RAT
96.1 Ubiquitin variant Ubv.21.4 P68202 RS27A_PLUXY
96.0 Ubiquitin variant Ubv.21.4 P62983 RS27A_MOUSE
96.1 Ubiquitin variant Ubv.21.4 P29504 RS27A_MANSE
96.0 Ubiquitin variant Ubv.21.4 P68200 RS27A_ICTPU
96.0 Ubiquitin variant Ubv.21.4 P62979 RS27A_HUMAN
96.1 Ubiquitin variant Ubv.21.4 P15357 RS27A_DROME
96.0 Ubiquitin variant Ubv.21.4 P79781 RS27A_CHICK
96.0 Ubiquitin variant Ubv.21.4 P62978 RS27A_CAVPO
96.0 Ubiquitin variant Ubv.21.4 P62992 RS27A_BOVIN
96.1 Ubiquitin variant Ubv.21.4 P0C276 RL40_SHEEP
96.1 Ubiquitin variant Ubv.21.4 P62986 RL40_RAT
96.1 Ubiquitin variant Ubv.21.4 P0C275 RL40_PONPY
96.1 Ubiquitin variant Ubv.21.4 P63053 RL40_PIG
96.1 Ubiquitin variant Ubv.21.4 P68205 RL40_OPHHA
96.1 Ubiquitin variant Ubv.21.4 P62984 RL40_MOUSE
96.1 Ubiquitin variant Ubv.21.4 P0C273 RL40_MACFA
96.1 Ubiquitin variant Ubv.21.4 P62987 RL40_HUMAN
96.1 Ubiquitin variant Ubv.21.4 P63052 RL40_FELCA
96.1 Ubiquitin variant Ubv.21.4 P18101 RL40_DROME
96.1 Ubiquitin variant Ubv.21.4 P63050 RL40_CANLF
96.1 Ubiquitin variant Ubv.21.4 P63048 RL40_BOVIN
95.0 Ubiquitin-specific protease 2 (USP2) O88623 UBP2_MOUSE
95.5 Ubiquitin-specific protease 2 (USP2) Q2KHV7 UBP2_BOVIN
95.5 Ubiquitin-specific protease 2 (USP2) Q5U349 UBP2_RAT
99.4 Ubiquitin-specific protease 2 (USP2) O75604 UBP2_HUMAN