PSMD5

INTERACTIONS
A schematic Model of the 19S RP Assembly and Roles of Base-Specific Chaperones
 PSMD5 acts as a chaperone during the assembly of the 26S proteasome,
specifically of the base subcomplex of the PA700/19S regulatory complex
(RC).
 In the initial step of the base subcomplex assembly is part of an
intermediate PSMD5:PSMC2:PSMC1:PSMD2 module which probably
assembles with a PSMD10:PSMC4:PSMC5:PAAF1 module followed by
dissociation of PSMD5.
 Base subunits, with the exception of Rpn2, form a complex in specific
combinations, where proteasome-dedicated chaperones directly interact
with specific ATPase subunits, thus shaping three distinct modules.
 While p28 and S5b positively regulate the association between the Rpt3-
Rpt6 complex and the Rpt1-Rpt2-Rpn1 complex, p27 inhibits the
association of the Rpt4-Rpt5 complex with the other two complexes.
 Accordingly, the assembly of the base subcomplex begins with the
association between the p28 module and S5b module, followed by
incorporation of the p27 module and Rpn2.
PSMC2 Domains-
InterPro domain ID-IPR003959, ATPase_AAA_core – 212-344
Pfam- PF00004, AAA – 212-344
InterPro domain ID-IPR041569, AAA_lid_3 – 367-411
InterPro domain ID-PF17862, AAA_lid_3 – 367-411
InterPro domain ID-IPR003593, AAA+_ATPase – 208-347
SMART-SM00382, AAA_5 – 208-347
PSMD5 Domains-

InterPro domain ID-IPR011989, ARM-like

InterPro domain ID-IPR016024, ARM-type_fold
PRALINE MSA of Hsm3 & PSMD5

Alignment score = 3600.00

Alignment score per
aligned residue pair = 7.68
Sequence identities = 53
Percent sequence identity = 0.11
Number of sequences = 2
Alignment length = 515
Number of residues = 984
Number of gaps = 46
PRALINE MSA of
Rpt1 & PSMC2
Alignment score = 6890.00
Alignment score per
aligned residue pair = 15.91
Sequence identities = 323
Percent sequence identity = 0.75
Number of sequences = 2
Alignment length = 467
Number of residues = 900
Number of gaps = 34
Interacting residues on the interacting faces of Hsm3 & Rpt1
 Rpt1- L-382, R-390, R-403, E-405, R-409, L-410, L-444, K-445, D-448,
S-452
 Hsm3- E-157, T-190, D-230, L-232
 Interactions is between α8, α10, α12 of Hsm3 & α2, α4, loop α1-2 of
Rpt1 by hydrogen bonding & van der Waals forces
 E-157 and T-190 of Hsm3 make a potential salt bridge and hydrogen
bond with Arg-409 and Arg-403, respectively.

C Terminal Domain of Rpt1 (381AA-467AA)

α1 α2 α4
L-382, R-390 R-403,E-405, R-409, L-410 L-444, K-445, D-448, S-452
MSA

Rpt1 PSMC2 Hsm3 PSMD5

L-382 L-382 E-157 I-157
R-390 K-390 T-190 T-190
R-403 R-403 D-230 D-230
E-405 E-405 L-232 L-232
R-409 R-409
L-410 L-410
L-444 L-444
K-445 E-445
D-448 N-448
S-452 K-452
E-157

R-409

Salt bridge between E-157 & R-409

 T-190

Hydrogen bond between R-403 & T-190

MSA of PSMD5 & Hsm3

sp|Q16401|PSMD5_HUMAN RVDLQRGLIHPDDSVKILTLSQIGRIVENSDAVTEILNNAELLKQIVYCIGGENLSVAKA 144

sp|P38348|HSM3_YEAST ENSQPKGLFATSNIIDIL-------------------------LDILFDEKVENDKLITA 155
. . :**: .: :.** :*:: ** .: .*
 
sp|Q16401|PSMD5_HUMAN AIKSLSRISLTQAGLEALFESNLL--DDLKSVMKTNDIVRYRVYELIIEI---SSVSPES 199
sp|P38348|HSM3_YEAST IEKALERLSTDELIRRRLFDNNLPYLVSVKGRMETVSFVRLIDFL-TIEFQFISGPEFKD 214
*:*.*:* : . **:.** .:*. *:* .:** : **: *. . :.
 
sp|Q16401|PSMD5_HUMAN LNYCTTSGLVTQLLRELTGEDVLVRATCIEMVTSLAY-THHGRQYLAQEGVIDQISNIIV 258
sp|P38348|HSM3_YEAST IIFCFTKEEILKSV-----EDILVFIELVNYYTKFLLEIRNQDKYWALRHV--------- 260
: :* *. : : : **:** :: *.: :: :* * . *
 
sp|Q16401|PSMD5_HUMAN GADSDPFSSFYLPGFVKFFGNLAVMDSPQQ---ICE-----RYPIFVEKVFEMIESQDPT 310
sp|P38348|HSM3_YEAST --------KKILPVFAQLFEDTENYPDVRAFSTNCLLQLFAEVSRIEEDEYSLFKTMDK- 311
. ** *.::* : . : * . : *. :.:::: *
PSMC2 & Rpt1 MSA
sp|P33299|PRS7_YEAST KKACIIFFDEIDAVGGARFDDGAGGDNEVQRTMLELITQLDGFDPRGNIKVMFATNRPNT360
sp|P35998|PRS7_HUMAN KKACLIFFDEIDAIGGARFDDGAGGDNEVQRTMLELINQLDGFDPRGNIKVLMATNRPDT326
****:********:***********************.*************::*****:*
 
sp|P33299|PRS7_YEAST LDPALLRPGRIDRKVEFSLPDLEGRANIFRIHSKSMSVERGIRWELISRLCPNSTGAELR420
sp|P35998|PRS7_HUMAN LDPALMRPGRLDRKIEFSLPDLEGRTHIFKIHARSMSVERDIRFELLARLCPNSTGAEIR386
*****:****:***:**********::**:**::******.**:**::**********:*
 
sp|P33299|PRS7_YEAST SVCTEAGMFAIRARRKVATEKDFLKAVDKVISGYKKFSSTSRYMQYN 467
sp|P35998|PRS7_HUMAN SVCTEAGMFAIRARRKIATEKDFLEAVNKVIKSYAKFSATPRYMTYN 433
****************:*******:**:***..* ***:* *** **
MSA of Hsm3, PSMD5, PSMC2, Rpt1
sp|P33299|PRS7_YEAST ------------DLEGRANIFRIHSKSMSVERGIRWELISRLCPNSTGAELRSVCTEAGM 428
sp|P35998|PRS7_HUMAN ------------DLEGRTHIFKIHARSMSVERDIRFELLARLCPNSTGAEIRSVCTEAGM 394
sp|Q16401|PSMD5_HUMAN TDDLLRMTESWFSSLSRD--------PLELFRGIS---------SQPFPEL--HCAALKV 417
sp|P38348|HSM3_YEAST ----AKLITEWLELINPQYLVKYHKDVVENYFHVS---------GYSIGMLRNLSADEEC 366
. . :. : . : .:
sp|P33299|PRS7_YEAST FAIRARRKVATEKDFL-----KAV-DKVI-----SGYKKFSSTSRYMQ------------ 465
sp|P35998|PRS7_HUMAN FAIRARRKIATEKDFL-----EAV-NKVI-----KSYAKFSATPRYMT------------ 431
sp|Q16401|PSMD5_HUMAN FTAIANQPWAQKLMFNSPGFVEYVVDRSVEHDKASKDAKYELVKALA---NSKTIAEIFG 474
sp|P38348|HSM3_YEAST FNAIRNK-FSAEIVLRLPYLEQMQVVET--------LTRYEYTSKFLLNEMPKVMGSLIG 417
* .: : : : : . ::. .
sp|P33299|PRS7_YEAST --------------YN-------------------------------------------- 467
sp|P35998|PRS7_HUMAN --------------YN-------------------------------------------- 433
sp|Q16401|PSMD5_HUMAN NPN-----------Y--LRLRTYLSEGP----YYVKPVS---TTAVEGAE---------- 504
sp|P38348|HSM3_YEAST DGSAGAIIDLETVHYRNSALRNLLDKGEEKLSVWYEPLLREYSKAVNGKNYSTGSETKIA 477
*
3VLD Crystal structure of yeast proteasome interacting protein
Crystalization Experiments
VAPOR DIFFUSION, HANGING DROP, 7pH, 293K,
8% PEG 3000, 0.2M potassium phosphate, 0.1M Tris-HCl, pH 7.0,
VAPOR DIFFUSION, HANGING DROP, temperature 293.0K
a = 85.402, b = 94.676, c = 129.19, α = 90, β = 90, γ = 90
Experimental Data
•Method: X-RAY DIFFRACTION
•Resolution: 2.05 Å
•R-Value Free: 0.240 
•R-Value Work: 0.186 
•R-Value Observed: 0.189 
•Space Group: P 21 21 21
•Organism(s): Saccharomyces cerevisiae S288C
•Expression System: Escherichia coli
3VLF Crystal structure of yeast proteasome interacting
protein
Crystalization Experiments
VAPOR DIFFUSION, HANGING DROP, 8.5pH, 277K, 1M
sodium chloride, 0.5% PEG 3350, 0.8M lithium sulfate,
0.1M N-(2-acetamido) iminodiacetic acid, pH 8.5, VAPOR
DIFFUSION, HANGING DROP, temperature 277K
a = 187.282, b = 187.282, c = 379.574, α = 90, β = 90, γ =
120
Experimental Data
•Method: X-RAY DIFFRACTION
•Resolution: 3.80 Å
•R-Value Free: 0.278 
•R-Value Work: 0.251 
•R-Value Observed: 0.251 
•Space Group: P 65 2 2
•Organism(s): Saccharomyces cerevisiae S288C
Expression System: Escherichia coli
3VLE Crystal structure of yeast proteasome interacting
protein
Crystalization Experiments
VAPOR DIFFUSION, HANGING DROP, 7pH, 293K,
8% PEG 3000, 0.2M potassium phosphate, 0.1M Tris-HCl,
pH 7.0,
VAPOR DIFFUSION, HANGING DROP, temperature 293.0K
a = 78.801, b = 91.893, c = 78.949, α = 90, β = 113.06, γ = 90
Experimental Data
•Method: X-RAY DIFFRACTION
•Resolution: 2.41 Å
•R-Value Free: 0.274 
•R-Value Work: 0.206 
•R-Value Observed: 0.209
•Organism(s): Saccharomyces cerevisiae S288C
•Expression System: Escherichia coli
4JPO 5A resolution structure of Proteasome Assembly Chaperone
Hsm3 in complex with a C-terminal fragment of Rpt1
Crystalization Experiments
VAPOR DIFFUSION, HANGING DROP, 7pH, 293K,
3M Sodium Formate, 100 mM Bis-Tris, 10mM
Calcium Chloride, pH 7.0, vapor diffusion, temperature 293K
a = 185.299, b = 185.299, c = 357.382, α = 90, β = 90, γ = 120
Experimental Data
•Method: X-RAY DIFFRACTION
•Resolution: 5.00 Å
•R-Value Free: 0.273 
•R-Value Work: 0.253 
•R-Value Observed: 0.254 
•Space Group: P 65 2 2
•Organism(s): Saccharomyces cerevisiae S288C
•Expression System: Escherichia coli BL21(DE3)
4FP7 2.2A resolution structure of Proteasome
Assembly Chaperone Hsm3
Crystalization Experiments
VAPOR DIFFUSION, 7pH, 293K,
20% PEG 4000, 100 mM Hepes,
150 mM ammonium sulfate, pH 7.0,
vapor diffusion, temperature 293K
a = 85.148, b = 94.466, c = 129.931, α = 90,
β = 90, γ = 90
Experimental Data
•Method: X-RAY DIFFRACTION
•Resolution: 2.20 Å
•R-Value Free: 0.239 
•R-Value Work: 0.188 
•R-Value Observed: 0.191 
•Space Group: P 21 21 21
•Organism(s): Saccharomyces cerevisiae S288C
•Expression System: Escherichia coli BL21(DE3)
4A3V yeast regulatory particle proteasome assembly
chaperone Hsm3 in complex with Rpt1 C-terminal fragment
Crystalization Experiments
VAPOR DIFFUSION, 7.5pH,
5M SODIUM FORMATE, 0.1M HEPES PH 7.5
a = 186.66, b = 186.66, c = 373.77, α = 90,
β = 90, γ = 120
Experimental Data Snapshot
•Method: X-RAY DIFFRACTION
•Resolution: 3.80 Å
•R-Value Free: 0.208 
•R-Value Work: 0.190 
•R-Value Observed: 0.191 
•Space Group: P 65 2 2
•Organism(s): Saccharomyces cerevisiae W303
•Expression System: Escherichia coli BL21
4A3T yeast regulatory particle proteasome
assembly chaperone Hsm3
Crystalization Experiments
VAPOR DIFFUSION,5.5pH,
20% PEG 4000, 0.2 M MALATE IMIDAZOLE, PH 5.5.
a = 86.07, b = 94.95, c = 129.95, α = 90,
β = 90, γ = 90
Experimental Data
•Method: X-RAY DIFFRACTION
•Resolution: 2.10 Å
•R-Value Free: 0.219 
•R-Value Work: 0.185 
•R-Value Observed: 0.186 
•Space Group: P 21 21 21
•Organism(s): Saccharomyces cerevisiae W303
•Expression System: Escherichia coli BL21
 PSMD5 gradient PCR

Temperature – 55 C, 56.2 C, 59.7 C, 64.3 C, 67.7 C, 69.0 C

PSMD5 large scale PCR amplification at 67.7 C & 69 C
Reactions – 2
Reaction vol – 100uL
PSMD5 large scale PCR amplification at 69 C
Reactions – 2
Reaction vol – 50uL