PDBsum entry 1lna

Metalloprotease

PDB id: 1lna

Contents

Protein chain

316 a.a. *

Ligands

VAL-LYS

DMS

Metals

_CO ×2

_CA ×3

Waters ×158

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Structural analysis of zinc substitutions in the active site of thermolysin.

Authors

D.R.Holland, A.C.Hausrath, D.Juers, B.W.Matthews.

Ref.

Protein Sci, 1995, 4, 1955-1965. [DOI no: 10.1002/pro.5560041001]

PubMed id

8535232

Abstract

Native thermolysin binds a single catalytically essential zinc ion that is tetrahedrally coordinated by three protein ligands and a water molecule. During catalysis the zinc ligation is thought to change from fourfold to fivefold. Substitution of the active-site zinc with Cd2+, Mn2+, Fe2+, and Co2+ alters the catalytic activity (Holmquist B, Vallee BL, 1974, J Biol Chem 249:4601-4607). Excess zinc inhibits the enzyme. To investigate the structural basis of these changes in activity, we have determined the structures of a series of metal-substituted thermolysins at 1.7-1.9 A resolution. The structure of the Co(2+)-substituted enzyme is shown to be very similar to that of wild type except that two solvent molecules are liganded to the metal at positions that are thought to be occupied by the two oxygens of the hydrated scissile peptide in the transition state. Thus, the enhanced activity toward some substrates of the cobalt-relative to the zinc-substituted enzyme may be due to enhanced stabilization of the transition state. The ability of Zn2+ and Co2+ to accept tetrahedral coordination in the Michaelis complex, as well as fivefold coordination in the transition state, may also contribute to their effectiveness in catalysis. The Cd(2+)- and Mn(2+)-substituted thermolysins display conformational changes that disrupt the active site to varying degrees and could explain the associated reduction of activity. The conformational changes involve not only the essential catalytic residue, Glu 143, but also concerted side-chain rotations in the adjacent residues Met 120 and Leu 144. Some of these side-chain movements are similar to adjustments that have been observed previously in association with the "hinge-bending" motion that is presumed to occur during catalysis by the zinc endoproteases. In the presence of excess zinc, a second zinc ion is observed to bind at His 231 within 3.2 A of the zinc bound to native thermolysin, explaining the inhibitory effect.

Secondary reference #1

Title

Structural comparison suggests that thermolysin and related neutral proteases undergo hinge-Bending motion during catalysis.

Authors

D.R.Holland, D.E.Tronrud, H.W.Pley, K.M.Flaherty, W.Stark, J.N.Jansonius, D.B.Mckay, B.W.Matthews.

Ref.

Biochemistry, 1992, 31, 11310-11316. [DOI no: 10.1021/bi00161a008]

PubMed id

1445869

Secondary reference #2

Title

Structure of thermolysin refined at 1.6 a resolution.

Authors

M.A.Holmes, B.W.Matthews.

Ref.

J Mol Biol, 1982, 160, 623-639. [DOI no: 10.1016/0022-2836(82)90319-9]

PubMed id

7175940

Figure 3.
FIG. 3. Conformational diagram for the backbone of thermolysin. Residues that are outside the ``allowed'' regions for a hard-sphere model are numbered.

Figure 4.
FIG. 4. Stereo diagram illustrating the apparent thermal motion of t,he thermolysin molecule. Larger circles correspond to residues with greater apparen motion. The radius of each c~wlr l\as obtained 1)~ taking the verage R value for all atoms in that residue, subtracting a constant value of 4.0 AZ (in order to make differences in apparent motion more obvious) and drawing the circle at t,hr SO'?; probabilit? level (Johson, 196.5).

The above figures are reproduced from the cited reference with permission from Elsevier

Secondary reference #3

Title

Structure of a mercaptan-Thermolysin complex illustrates mode of inhibition of zinc proteases by substrate-Analogue mercaptans.

Authors

A.F.Monzingo, B.W.Matthews.

Ref.

Biochemistry, 1982, 21, 3390-3394. [DOI no: 10.1021/bi00257a022]

PubMed id

7052122

Secondary reference #4

Title

Binding of hydroxamic acid inhibitors to crystalline thermolysin suggests a pentacoordinate zinc intermediate in catalysis.

Authors

M.A.Holmes, B.W.Matthews.

Ref.

Biochemistry, 1981, 20, 6912-6920. [DOI no: 10.1021/bi00527a026]

PubMed id

7317361

Secondary reference #5

Title

Binding of the biproduct analog l-Benzylsuccinic acid to thermolysin determined by X-Ray crystallography.

Authors

M.C.Bolognesi, B.W.Matthews.

Ref.

J Biol Chem, 1979, 254, 634-639.

PubMed id

762086

Secondary reference #6

Title

Comparison of the structures of carboxypeptidase a and thermolysin.

Authors

W.R.Kester, B.W.Matthews.

Ref.

J Biol Chem, 1977, 252, 7704-7710.

PubMed id

914833

Secondary reference #7

Title

A crystallographic study of the complex of phosphoramidon with thermolysin. A model for the presumed catalytic transition state and for the binding of extended substances.

Authors

L.H.Weaver, W.R.Kester, B.W.Matthews.

Ref.

J Mol Biol, 1977, 114, 119-132.

PubMed id

909082

Secondary reference #8

Title

Crystallographic study of the binding of dipeptide inhibitors to thermolysin: implications for the mechanism of catalysis.

Authors

W.R.Kester, B.W.Matthews.

Ref.

Biochemistry, 1977, 16, 2506-2516. [DOI no: 10.1021/bi00630a030]

PubMed id

861218

Secondary reference #9

Title

Role of calcium in the thermal stability of thermolysin.

Authors

F.W.Dahlquist, J.W.Long, W.L.Bigbee.

Ref.

Biochemistry, 1976, 15, 1103-1111. [DOI no: 10.1021/bi00650a024]

PubMed id

814920

Secondary reference #10

Title

Evidence of homologous relationship between thermolysin and neutral protease a of bacillus subtilis

Authors

P.L.Levy, M.K.Pangburn, Y.Burstein, L.H.Ericsson, H.Neurath, K.A.Walsh.

Ref.

atlas of protein sequence, 1976, 5, 98.

Secondary reference #11

Title

The conformation of thermolysin.

Authors

B.W.Matthews, L.H.Weaver, W.R.Kester.

Ref.

J Biol Chem, 1974, 249, 8030-8044.

PubMed id

4214815

Secondary reference #12

Title

Binding of lanthanide ions to thermolysin.

Authors

B.W.Matthews, L.H.Weaver.

Ref.

Biochemistry, 1974, 13, 1719-1725. [DOI no: 10.1021/bi00705a025]

PubMed id

4831359

Secondary reference #13

Title

The structure of thermolysin: an electron density map at 2-3 a resolution.

Authors

P.M.Colman, J.N.Jansonius, B.W.Matthews.

Ref.

J Mol Biol, 1972, 70, 701-724. [DOI no: 10.1016/0022-2836(72)90569-4]

PubMed id

5083153

Figure 5.
Fra. 5. Schematic diagram, seen from above, of the optical comparator used to build to thermolyein model after Richards, 1968).

Figure 9.
FIG. 9. (a) Key showing tho position of the major binding sites of Llra heavy BWIUS used to determirw thn phase trnglaa fur L~IC therrnolysin elootron density map. and for the folluwing diEcmnoe maps. The calcum positions LIY dotcnninod from tho throo -dinmtional map BK+ indicated in this Figure by crosses. DA&IA, imnrcury acetic acd. (b) DifYeronce electron density bdween atrontillm-thnrmolynin and native themolysin. Tho mnoutmn of this and the following maps are 2-4 L% (c) DifGrence electron den&y betweau barium-lborxnolyuin au3 uetivo thermolyti. (d) Diffeence obctro density between dysprosium-thermolyain and native thomolysin.

The above figures are reproduced from the cited reference with permission from Elsevier

Secondary reference #14

Title

Amino-Acid sequence of thermolysin

Authors

K.Titani, M.A.Hermodson, L.H.Ericsson, K.A.Walsh, H.Neurath.

Ref.

nature new biol, 1972, 238, 35.

Secondary reference #15

Title

Three dimensional structure of thermolysin

Authors

B.W.Matthews, J.N.Jansonius, P.M.Colman, B.P.Schoenborn, D.Duporque.

Ref.

nature new biol, 1972, 238, 37.

Secondary reference #16

Title

Structure of thermolysin

Authors

B.W.Matthews, P.M.Colman, J.N.Jansonius, K.Titani, K.A.Walsh, H.Neurath.

Ref.

nature new biol, 1972, 238, 41.