Trade-offs between enzyme fitness and solubility illuminated by deep mutational scanning.


Abstract

Proteins are marginally stable, and an understanding of the sequence determinants for improved protein solubility is highly desired. For enzymes, it is well known that many mutations that increase protein solubility decrease catalytic activity. These competing effects frustrate efforts to design and engineer stable, active enzymes without laborious high-throughput activity screens. To address the trade-off between enzyme solubility and activity, we performed deep mutational scanning using two different screens/selections that purport to gauge protein solubility for two full-length enzymes. We assayed a TEM-1 beta-lactamase variant and levoglucosan kinase (LGK) using yeast surface display (YSD) screening and a twin-arginine translocation pathway selection. We then compared these scans with published experimental fitness landscapes. Results from the YSD screen could explain 37% of the variance in the fitness landscapes for one enzyme. Five percent to 10% of all single missense mutations improve solubility, matching theoretical predictions of global protein stability. For a given solubility-enhancing mutation, the probability that it would retain wild-type fitness was correlated with evolutionary conservation and distance to active site, and anticorrelated with contact number. Hybrid classification models were developed that could predict solubility-enhancing mutations that maintain wild-type fitness with an accuracy of 90%. The downside of using such classification models is the removal of rare mutations that improve both fitness and solubility. To reveal the biophysical basis of enhanced protein solubility and function, we determined the crystallographic structure of one such LGK mutant. Beyond fundamental insights into trade-offs between stability and activity, these results have potential biotechnological applications.

Submission Details

ID: FpUWknYT

Submitter: Connie Wang

Submission Date: Oct. 26, 2018, 2:33 p.m.

Version: 1

Publication Details
Klesmith JR;Bacik JP;Wrenbeck EE;Michalczyk R;Whitehead TA,Proc Natl Acad Sci U S A (2017) Trade-offs between enzyme fitness and solubility illuminated by deep mutational scanning. PMID:28196882
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
1M40 2002-07-17 0.85 ULTRA HIGH RESOLUTION CRYSTAL STRUCTURE OF TEM-1
4ID4 2013-12-25 1.05 Crystal structure of chimeric beta-lactamase cTEM-17m
4R4S 2015-11-11 1.1 Crystal structure of chimeric beta-lactamase cTEM-19m at 1.1 angstrom resolution
4QY6 2015-08-12 1.15 Crystal structures of chimeric beta-lactamase cTEM-19m showing different conformations
1NYM 2003-08-26 1.2 Crystal Structure of the complex between M182T mutant of TEM-1 and a boronic acid inhibitor (CXB)
4R4R 2015-11-11 1.2 Crystal structure of chimeric beta-lactamase cTEM-19m at 1.2 angstrom resolution
4RX3 2015-03-04 1.39 A triple mutant in the omega-loop of TEM-1 beta-lactamase changes the substrate profile via a large conformational change and an altered general base for catalysis
1YT4 2005-07-12 1.4 Crystal structure of TEM-76 beta-lactamase at 1.4 Angstrom resolution
5HW5 2017-06-28 1.41 Crystal structure of TEM1 beta-lactamase in the presence of 2.0 MPa xenon
4RVA 2015-03-04 1.44 A triple mutant in the omega-loop of TEM-1 beta-lactamase changes the substrate profile via a large conformational change and an altered general base for deacylation
6AYK 2018-09-12 1.44 Crystal structure of TEM1 beta-lactamase mutant I263A in the presence of 1.2 MPa xenon
1PZP 2004-03-09 1.45 TEM-1 Beta-Lactamase in Complex with a Novel, Core-Disrupting, Allosteric Inhibitor
4QY5 2015-08-12 1.5 Crystal structures of chimeric beta-lactamase cTEM-19m showing different conformations
5KPU 2017-08-09 1.5 Crystal structure of TEM1 beta-lactamase mutant I263L in the presence of 1.2 MPa xenon
1LI9 2002-09-11 1.52 Crystal structure of TEM-34 beta-Lactamase at 1.5 Angstrom
4ZJ1 2015-05-20 1.54 Crystal Structure of p-acrylamido-phenylalanine modified TEM1 beta-lactamase from Escherichia coli : V216AcrF mutant
1ZG4 2005-05-17 1.55 TEM1 beta lactamase
1NXY 2003-08-26 1.6 Crystal Structure of the complex between M182T mutant of TEM-1 and a boronic acid inhibitor (SM2)
2V1Z 2008-06-24 1.6 Structure of a TEM-1 beta-lactamase insertant allosterically regulated by kanamycin and anions.
1LI0 2002-09-11 1.61 Crystal structure of TEM-32 beta-Lactamase at 1.6 Angstrom
5HVI 2017-06-28 1.64 Crystal structure of TEM1 beta-lactamase
2B5R 2006-04-11 1.65 1B Lactamase / B Lactamase Inhibitor
2V20 2008-06-24 1.67 Structure of a TEM-1 beta-lactamase insertant allosterically regulated by kanamycin and anions. Complex with sulfate.
1ERM 2000-05-10 1.7 X-RAY CRYSTAL STRUCTURE OF TEM-1 BETA LACTAMASE IN COMPLEX WITH A DESIGNED BORONIC ACID INHIBITOR (1R)-1-ACETAMIDO-2-(3-CARBOXYPHENYL)ETHANE BORONIC ACID
4ZJ3 2015-05-20 1.7 Crystal structure of cephalexin bound acyl-enzyme intermediate of Val216AcrF mutant TEM1 beta-lactamase from Escherichia coli: E166N and V216AcrF mutant.
5HW1 2017-06-28 1.7 Crystal structure of TEM1 beta-lactamase in the presence of 1.2 MPa xenon
1FQG 2000-11-01 1.7 MOLECULAR STRUCTURE OF THE ACYL-ENZYME INTERMEDIATE IN TEM-1 BETA-LACTAMASE
1JTG 2001-10-17 1.73 CRYSTAL STRUCTURE OF TEM-1 BETA-LACTAMASE / BETA-LACTAMASE INHIBITOR PROTEIN COMPLEX
1JVJ 2002-03-06 1.73 CRYSTAL STRUCTURE OF N132A MUTANT OF TEM-1 BETA-LACTAMASE IN COMPLEX WITH A N-FORMIMIDOYL-THIENAMYCINE
1JWP 2002-06-05 1.75 Structure of M182T mutant of TEM-1 beta-lactamase
1NY0 2003-08-26 1.75 Crystal Structure of the complex between M182T mutant of TEM-1 and a boronic acid inhibitor (NBF)
5I52 2017-06-28 1.75 Crystal structure of TEM1 beta-lactamase mutant I263N
1JWZ 2002-06-05 1.8 Crystal structure of TEM-64 beta-lactamase in complex with a boronic acid inhibitor (105)
1BT5 1999-09-02 1.8 CRYSTAL STRUCTURE OF THE IMIPENEM INHIBITED TEM-1 BETA-LACTAMASE FROM ESCHERICHIA COLI
1BTL 1995-01-26 1.8 CRYSTAL STRUCTURE OF ESCHERICHIA COLI TEM1 BETA-LACTAMASE AT 1.8 ANGSTROMS RESOLUTION
4ZJ2 2015-05-20 1.8 Crystal Structure of p-acrylamido-phenylalanine modified TEM1 beta-lactamase from Escherichia coli :E166N mutant
5KKF 2017-06-28 1.82 Crystal structure of TEM1 beta-lactamase mutant I263L
1JWV 2002-06-05 1.85 Crystal structure of G238A mutant of TEM-1 beta-lactamase in complex with a boronic acid inhibitor (sefb4)
6APA 2018-08-22 1.86 Crystal structure of TEM1 beta-lactamase mutant I263A
1XPB 1997-04-01 1.9 STRUCTURE OF BETA-LACTAMASE TEM1
1ERQ 2000-05-10 1.9 X-RAY CRYSTAL STRUCTURE OF TEM-1 BETA LACTAMASE IN COMPLEX WITH A DESIGNED BORONIC ACID INHIBITOR (1R)-1-ACETAMIDO-2-(3-CARBOXY-2-HYDROXYPHENYL)ETHYL BORONIC ACID
1PZO 2004-03-09 1.9 TEM-1 Beta-Lactamase in Complex with a Novel, Core-Disrupting, Allosteric Inhibitor
3TOI 2012-05-16 1.9 Tailoring Enzyme Stability and Exploiting Stability-Trait Linkage by Iterative Truncation and Optimization
1XXM 2005-01-18 1.9 The modular architecture of protein-protein binding site
1NYY 2003-08-26 1.9 Crystal Structure of the complex between M182T mutant of TEM-1 and a boronic acid inhibitor (105)
4GKU 2012-10-10 1.91 Crystal structure of beta lactamase in PET-15B
3CMZ 2008-11-25 1.92 TEM-1 Class-A beta-lactamase L201P mutant apo structure
5NPO 2017-12-20 1.95 Promiscuous Protein Self-Assembly as a Function of Protein Stability
1TEM 1997-05-15 1.95 6 ALPHA HYDROXYMETHYL PENICILLOIC ACID ACYLATED ON THE TEM-1 BETA-LACTAMASE FROM ESCHERICHIA COLI
5I63 2017-06-28 1.95 Crystal structure of TEM1 beta-lactamase mutant I263N in the presence of 1.2 MPa xenon
3DTM 2008-08-05 2.0 Increased folding stability of TEM-1 beta-lactamase by in-vitro selection
6B2N 2018-01-17 2.0 Crystal structure of TEM-1 beta-lactamase mutant M182N
1LHY 2002-09-11 2.0 Crystal structure of TEM-30 beta-Lactamase at 2.0 Angstrom
1AXB 1998-10-28 2.0 TEM-1 BETA-LACTAMASE FROM ESCHERICHIA COLI INHIBITED WITH AN ACYLATION TRANSITION STATE ANALOG
1ESU 2000-05-03 2.0 S235A MUTANT OF TEM1 BETA-LACTAMASE
4MEZ 2014-10-15 2.05 Crystal structure of M68L/M69T double mutant TEM-1
5IQ8 2017-06-28 2.06 Crystal structure of TEM1 beta-lactamase mutant A224C/G283C disulfide
3C7V 2008-10-07 2.07 Structural Insight into the Kinetics and Delta-Cp of interactions between TEM-1 Beta-Lactamase and BLIP
1ZG6 2005-05-17 2.1 TEM1 beta lactamase mutant S70G
1ERO 2000-05-10 2.1 X-RAY CRYSTAL STRUCTURE OF TEM-1 BETA LACTAMASE IN COMPLEX WITH A DESIGNED BORONIC ACID INHIBITOR (1R)-2-PHENYLACETAMIDO-2-(3-CARBOXYPHENYL)ETHYL BORONIC ACID
3C7U 2008-10-07 2.2 Structural Insight into the Kinetics and Cp of interactions between TEM-1-Lactamase and BLIP
4IBR 2013-04-03 2.2 Crystal structure of stabilized TEM-1 beta-lactamase variant v.13 carrying G238S/E104K mutations
1CK3 1999-08-18 2.28 N276D MUTANT OF ESCHERICHIA COLI TEM-1 BETA-LACTAMASE
4DXB 2012-08-08 2.29 2.29A structure of the engineered MBP TEM-1 fusion protein RG13 in complex with zinc, P1 space group
1S0W 2004-02-10 2.3 1b Lactamse/ b Lactamase Inhibitor
1JTD 2001-10-03 2.3 Crystal structure of beta-lactamase inhibitor protein-II in complex with TEM-1 beta-lactamase
4DXC 2012-08-08 2.3 Crystal structure of the engineered MBP TEM-1 fusion protein RG13, C2 space group
4RX2 2015-03-04 2.32 A triple mutant in the omega-loop of TEM-1 beta-lactamase changes the substrate profile via a large conformational change and an altered general base for catalysis
4IBX 2013-04-03 2.68 Crystal structure of stabilized TEM-1 beta-lactamase variant v.13
3JYI 2009-10-06 2.7 Structural and biochemical evidence that a TEM-1 {beta}-lactamase Asn170Gly active site mutant acts via substrate-assisted catalysis

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Beta-lactamase P62593 BLAT_ECOLX
100.0 Beta-lactamase P62594 BLAT_SALTI
99.7 Beta-lactamase Q48406 BLAT_KLEOX