Structure-based protein engineering efforts with a monomeric TIM variant: the importance of a single point mutation for generating an active site with suitable binding properties.


Abstract

A monomeric variant of triosephosphate isomerase (TIM) with a new engineered binding groove has been characterized further. In this variant (ml8bTIM), the phosphate binding loop had been shortened, causing the binding site to be much more extended. Here, it is reported that in the V233A variant of ml8bTIM (A-TIM), three important properties of the wild-type TIM active site have been restored: (i) the structural properties of loop-7, (ii) the binding site of a conserved water molecule between loop-7 and loop-8 and (iii) the binding site of the phosphate moiety. It is shown that the active site of A-TIM can bind TIM transition state analogs and suicide inhibitors competently. It is found that the active site geometry of the A-TIM complexes is less compact and more solvent exposed, as in wild-type TIM. This correlates with the observation that the catalytic efficiency of A-TIM for interconverting the TIM substrates is too low to be detected. It is also shown that the A-TIM active site can bind compounds which do not bind to wild-type TIM and which are completely different from the normal TIM substrate, like a citrate molecule. The binding of this citrate molecule is stabilized by hydrogen bonding interactions with the new binding groove. Study holds ProTherm entries: 24486, 24487 Extra Details: citrate; monomeric TIM; protein design; suicide inhibitor; transition state analog

Submission Details

ID: sEUbBhv6

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:55 p.m.

Version: 1

Publication Details
Alahuhta M;Salin M;Casteleijn MG;Kemmer C;El-Sayed I;Augustyns K;Neubauer P;Wierenga RK,Protein Eng. Des. Sel. (2008) Structure-based protein engineering efforts with a monomeric TIM variant: the importance of a single point mutation for generating an active site with suitable binding properties. PMID:18239072
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
1AG1 1997-03-28T00:00:00+0000 2.36 MONOHYDROGEN PHOSPHATE BINDING TO TRYPANOSOMAL TRIOSEPHOSPHATE ISOMERASE
1DKW 1999-12-08T00:00:00+0000 2.65 CRYSTAL STRUCTURE OF TRIOSE-PHOSPHATE ISOMERASE WITH MODIFIED SUBSTRATE BINDING SITE
1IIG 2001-04-23T00:00:00+0000 2.6 STRUCTURE OF TRYPANOSOMA BRUCEI BRUCEI TRIOSEPHOSPHATE ISOMERASE COMPLEXED WITH 3-PHOSPHONOPROPIONATE
1IIH 2001-04-23T00:00:00+0000 2.2 STRUCTURE OF TRYPANOSOMA BRUCEI BRUCEI TRIOSEPHOSPHATE ISOMERASE COMPLEXED WITH 3-PHOSPHOGLYCERATE
1KV5 2002-01-25T00:00:00+0000 1.65 Structure of Trypanosoma brucei brucei TIM with the salt-bridge-forming residue Arg191 mutated to Ser
1ML1 1996-09-27T00:00:00+0000 2.6 PROTEIN ENGINEERING WITH MONOMERIC TRIOSEPHOSPHATE ISOMERASE: THE MODELLING AND STRUCTURE VERIFICATION OF A SEVEN RESIDUE LOOP
1MSS 1994-07-27T00:00:00+0000 2.4 LARGE SCALE STRUCTURAL REARRANGEMENTS OF THE FRONT LOOPS IN MONOMERISED TRIOSEPHOSPHATE ISOMERASE, AS DEDUCED FROM THE COMPARISON OF THE STRUCTURAL PROPERTIES OF MONOTIM AND ITS POINT MUTATION VARIANT MONOSS
1TPD 1994-02-28T00:00:00+0000 2.1 STRUCTURES OF THE "OPEN" AND "CLOSED" STATE OF TRYPANOSOMAL TRIOSEPHOSPHATE ISOMERASE, AS OBSERVED IN A NEW CRYSTAL FORM: IMPLICATIONS FOR THE REACTION MECHANISM
1TPE 1994-02-28T00:00:00+0000 2.1 COMPARISON OF THE STRUCTURES AND THE CRYSTAL CONTACTS OF TRYPANOSOMAL TRIOSEPHOSPHATE ISOMERASE IN FOUR DIFFERENT CRYSTAL FORMS
1TPF 1994-02-28T00:00:00+0000 1.8 COMPARISON OF THE STRUCTURES AND THE CRYSTAL CONTACTS OF TRYPANOSOMAL TRIOSEPHOSPHATE ISOMERASE IN FOUR DIFFERENT CRYSTAL FORMS

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Triosephosphate isomerase, glycosomal P04789 TPIS_TRYBB