Single Residue Research Articles

In vivo functional characterization of EGFR variants identifies novel drivers of glioblastoma.

Glioblastoma is the most common and aggressive primary brain tumor. Large-scale sequencing initiatives have cataloged its mutational landscape in hopes of elucidating mechanisms driving this deadly disease. However, a major bottleneck in harnessing this data for new therapies is deciphering "driver" and "passenger" events amongst the vast volume of information. We utilized an autochthonous, in vivo screening approach to identify driver, EGFR variants. RNA-Seq identified unique molecular signatures of mouse gliomas across these variants, which only differ by a single amino acid change. In particular, we identified alterations to lipid metabolism, which we further validated through an unbiased lipidomics screen. Our screen identified A289I as the most potent EGFR variant, which has previously not been characterized. One of the mechanisms through which A289I promotes gliomagenesis is to alter cellular triacylglycerides through MTTP. Knockout of Mttp in mouse gliomas, reduces gliomagenesis in multiple models. EGFR variants that differ by a single amino acid residue differentially promote gliomagenesis. Among the identified mechanism that drives glioma growth include lipid metabolism through MTTP. Understanding triacylglyceride accumulation may present a prospective therapeutic pathway for this deadly disease.

Research into the Bioengineering of a Novel α-Conotoxin from the Milked Venom of Conus obscurus.

The marine cone snail produces one of the fastest prey strikes in the animal kingdom. It injects highly efficacious venom, often causing prey paralysis and death within seconds. Each snail has hundreds of conotoxins, which serve as a source for discovering and utilizing novel analgesic peptide therapeutics. In this study, we discovered, isolated, and synthesized a novel α3/5-conotoxins derived from the milked venom of Conus obscurus (α-conotoxin OI) and identified the presence of α-conotoxin SI-like sequence previously found in the venom of Conus striatus. Five synthetic analogs of the native α-conotoxin OI were generated. These analogs incorporated single residue or double residue mutations. Three synthetic post-translational modifications (PTMs) were synthetically incorporated into these analogs: N-terminal truncation, proline hydroxylation, and tryptophan bromination. The native α-conotoxin OI demonstrated nanomolar potency in Poecilia reticulata and Homo sapiens muscle-type nicotinic acetylcholine receptor (nAChR) isoforms. Moreover, the synthetic α-[P9K] conotoxin OI displayed enhanced potency in both bioassays, ranging from a 2.85 (LD50) to 18.4 (IC50) fold increase in comparative bioactivity. The successful incorporation of PTMs, with retention of both potency and nAChR isoform selectivity, ultimately pushes new boundaries of peptide bioengineering and the generation of novel α-conotoxin-like sequences.

Functional and structural characterization of Norovirus GII.6 in recognizing histo-blood group antigens

Noroviruses (NoVs) are the primary cause of acute gastroenteritis worldwide. Histo-blood group antigens (HBGAs) are receptors or attachment factors that affect the prevalence and host susceptibility of NoVs. GII.6 NoV is one of the predominant genotypes in humans, which recognizes the type ABO secretor of HBGAs. However, the structural basis of GII.6 NoV's interaction with HBGAs receptors remains elusive. In this study, we investigated the binding features of the GII.6 strain to HBGAs using saliva- and glycan-ELISA assays and characterized the molecular basis of the GII.6 virus that recognizes H disaccharide. We showed that the GII.6 ​P domain recognized some A and O secretor's saliva samples, most B secretor's saliva samples, and H disaccharide antigen, but did not bind non-secretors’ saliva. Further, we determined the crystal structures of GII.6 and its complex with H disaccharides at 1.7 ​Å, revealing that the P domain of GII.6 shares the conventional binding interface and mode of GII HBGAs. Single residue mutations at the GII.6-H binding sites could inhibit the binding of GII.6 to HBGAs, demonstrating that the interaction residues were crucial in maintaining NoV-glycan integrity. Finally, structural and sequence analyses showed that the major residues of the GII.6-H interaction were conserved among NoVs in the GII genogroup. Taken together, our study characterized the functional and structural features of GII.6 that allow it to interact with HBGAs, and shed light on NoV evolution, epidemiology, and anti-viral drug development.

PREPARATION AND APPLICATION OF CHITOSAN/ATTAPULGITE COMPOSITE FLOCCULANT

A chitosan/attapulgite (CS/ATP) composite flocculant with excellent adsorption was prepared from chitosan (CS) and attapulgite clay (ATP). The micro morphology and structure of the composite flocculant were characterized with SEM and IR. The effect of the CS/ATP composite flocculant on the recovery of the main components from the bezafibrate (BZ) wastewater of a pharmaceutical plant was investigated. The influences of the flocculation temperature, mixing time and standing time on the flocculation effect were investigated by determining the BZ content of the recovered crude products as the evaluation index so as to obtain the best recovery process. The experimental results show that the best flocculation condition is created when we add 20 mg/mL of CS/ATP to the waste liquid, stir it at 30 °C for 10 min and allow it to stand for 30 min. Compared with the traditional recovery process, the BZ content of the recovered crude products can be increased from 47 % to 74 % when using the CS/ATP composite flocculant. This flocculant can effectively overcome the defects of single material residues and difficult separation, accelerate the settlement of impurities in a waste liquid effectively, and can be used in the field of solution clearing and waste liquid separation.

Site-Selective Lysine Acetylation of Human Immunoglobulin G for Immunoliposomes and Bispecific Antibody Complexes.

Site-selective acetylation of a single lysine residue in a protein that reaches a lysine acetyltransferase's accuracy, precision, and reliability is challenging. Here, we report a peptide-guided, proximity-driven group transfer reaction that acetylates a single lysine residue, Lys 248, of the fragment crystallizable region (Fc region) in the heavy chain of the human Immunoglobulin G (IgG). An Fc-interacting peptide bound with the Fc domain and positioned a phenolic ester close to Lys 248, which induced a nucleophilic reaction and resulted in the transfer of an acetyl group to Lys 248. The acetylation reaction proceeded to a decent yield under the physiological condition without the need for deglycosylation, unnatural amino acids, or catalysts. Along with acetylation, functional moieties such as azide, alkyne, fluorescent molecules, or biotin could also be site-selectively installed on Lys 248, allowing IgG's further derivatization. We then synthesized an antibody-lipid conjugate and constructed antibody-conjugated liposomes (immunoliposomes), targeting HER2-positive (HER2+) cancer cells. We also built a bispecific antibody complex (bsAbC) covalently linking an anti-HER2 antibody and an anti-CD3 antibody. The bsAbC showed in vitro effector-cell-mediated cytotoxicity at nanomolar concentrations. Compared with bispecific antibodies (bsAbs), bsAbCs are constructed based on native IgGs and contain two antigen-binding sites to each antigen, twice that of bsAbs. Altogether, this work reports a method of site-selective acetylation of native antibodies, highlights a facile way of site-selective IgG functionalization, and underscores the potential of bsAbCs in cancer immunotherapy.

Structural basis for the interaction between the first SURP domain of the SF3A1 subunit in U2 snRNP and the human splicing factor SF1.

SURP domains are exclusively found in splicing‐related proteins in all eukaryotes. SF3A1, a component of the U2 snRNP, has two tandem SURP domains, SURP1, and SURP2. SURP2 is permanently associated with a specific short region of SF3A3 within the SF3A protein complex whereas, SURP1 binds to the splicing factor SF1 for recruitment of U2 snRNP to the early spliceosomal complex, from which SF1 is dissociated during complex conversion. Here, we determined the solution structure of the complex of SURP1 and the human SF1 fragment using nuclear magnetic resonance (NMR) methods. SURP1 adopts the canonical topology of α1–α2–310–α3, in which α1 and α2 are connected by a single glycine residue in a particular backbone conformation, allowing the two α‐helices to be fixed at an acute angle. A hydrophobic patch, which is part of the characteristic surface formed by α1 and α2, specifically contacts a hydrophobic cluster on a 16‐residue α‐helix of the SF1 fragment. Furthermore, whereas only hydrophobic interactions occurred between SURP2 and the SF3A3 fragment, several salt bridges and hydrogen bonds were found between the residues of SURP1 and the SF1 fragment. This finding was confirmed through mutational studies using bio‐layer interferometry. The study also revealed that the dissociation constant between SURP1 and the SF1 fragment peptide was approximately 20 μM, indicating a weak or transient interaction. Collectively, these results indicate that the interplay between U2 snRNP and SF1 involves a transient interaction of SURP1, and this transient interaction appears to be common to most SURP domains, except for SURP2.

The dynamic interplay of PIP2 and ATP in the regulation of the KATP channel.

ATP-sensitive potassium (KATP ) channels couple the intracellular ATP concentration to insulin secretion. KATP channel activity is inhibited by ATP binding to the Kir6.2 tetramer and activated by phosphatidylinositol 4,5-bisphosphate (PIP2 ). Here, we use molecular dynamics simulation, electrophysiology and fluorescence spectroscopy to show that ATP and PIP2 occupy different binding pockets that share a single amino acid residue, K39. When both ligands are present, simulations suggest that K39 shows a greater preference to co-ordinate with PIP2 than with ATP. They also predict that a neonatal diabetes mutation at K39 (K39R) increases the number of hydrogen bonds formed between K39 and PIP2 , potentially accounting for the reduced ATP inhibition observed in electrophysiological experiments. Our work suggests that PIP2 and ATP interact allosterically to regulate KATP channel activity. KEY POINTS: The KATP channel is activated by the binding of phosphatidylinositol 4,5-bisphosphate (PIP2 ) lipids and inactivated by the binding of ATP. K39 has the potential to bind to both PIP2 and ATP. A mutation to this residue (K39R) results in neonatal diabetes. This study uses patch-clamp fluorometry, electrophysiology and molecular dynamics simulation. We show that PIP2 competes with ATP for K39, and this reduces channel inhibition by ATP. We show that K39R increases channel affinity to PIP2 by increasing the number of hydrogen bonds with PIP2 , when compared with the wild-type K39. This therefore decreases KATP channel inhibition by ATP.

Conformational stability of ageritin, a metal binding ribotoxin-like protein of fungal origin

Ageritin is a ribotoxin-like protein of biotechnological interest, belonging to a family of ribonucleases from edible mushrooms. Its enzymatic activity is explicated through the hydrolysis of a single phosphodiester bond, located in the sarcin/ricin loop of ribosomes. Unlike other ribotoxins, ageritin activity requires divalent cations (Zn2+). Here we investigated the conformational stability of ageritin in the pH range 4.0–7.4, using calorimetric and spectroscopic techniques. We observed a high protein thermal stability at all pHs with a denaturation temperature of 78 °C. At pH 5.0 we calculated a value of 36 kJ mol−1 for the unfolding Gibbs energy at 25 °C. We also analysed the thermodynamic and catalytic behaviour of S-pyridylethylated form, obtained by alkylating the single Cys18 residue, which is predicted to bind Zn2+. We show that this form possesses the same activity and structure of ageritin, but lower stability. In fact, the corresponding values of 52 °C and 14 kJ mol−1 were found. Conservation of activity is consistent with the location of alkylation site on the opposite site of the catalytic site cleft. Inasmuch as Cys18 is part of a structurally stabilizing zinc-binding site, disrupted by cysteine alkylation, our results point to an important role of metal ions in ageritin stability.

Characterization of adjacent charged residues near the agonist binding site of the nematode UNC-49 GABA receptor

The UNC-49 receptor is a Cys-loop GABA receptor that is unique to the nematode phylum. The receptor differs from mammalian GABA receptors both in amino acid sequence and pharmacology which highlights its potential as a novel anthelmintic target. Sequence differences within and near the various ligand-binding loops of the nematode receptor suggest that there could be structural differences compared to mammalian receptors that result in different pharmacological and functional features. Here we investigated three residues in the UNC-49 receptor from the parasitic nematode Haemonchus contortus: K181, E183, and T230. Analysis of these residues was conducted via site-directed mutagenesis, electrophysiology, MD simulations, and mutant cycling analysis. In the UNC-49 receptor, E183 lies in close proximity to K181 where together they appear to play a role in GABA sensitivity and pharmacology, possibly interacting via an ionic bond. While the introduction of single alanine residues at each position separately had a negative impact on GABA EC50, the double alanine mutant (K181A/E183A) exhibited wildtype-level GABA EC50 and some differences in pharmacology. Overall, this study has revealed a potentially novel role for these two residues in nematode UNC-49 GABA receptors that could aid in understanding their function.

Role of the reaction-structure coupling in temperature compensation of the KaiABC circadian rhythm.

When the mixture solution of cyanobacterial proteins, KaiA, KaiB, and KaiC, is incubated with ATP in vitro, the phosphorylation level of KaiC shows stable oscillations with the temperature-compensated circadian period. Elucidating this temperature compensation is essential for understanding the KaiABC circadian clock, but its mechanism has remained a mystery. We analyzed the KaiABC temperature compensation by developing a theoretical model describing the feedback relations among reactions and structural transitions in the KaiC molecule. The model showed that the reduced structural cooperativity should weaken the negative feedback coupling among reactions and structural transitions, which enlarges the oscillation amplitude and period, explaining the observed significant period extension upon single amino-acid residue substitution. We propose that an increase in thermal fluctuations similarly attenuates the reaction-structure feedback, explaining the temperature compensation in the KaiABC clock. The model explained the experimentally observed responses of the oscillation phase to the temperature shift or the ADP-concentration change and suggested that the ATPase reactions in the CI domain of KaiC affect the period depending on how the reaction rates are modulated. The KaiABC clock provides a unique opportunity to analyze how the reaction-structure coupling regulates the system-level synchronized oscillations of molecules.

Quantum chemistry study of the multiphoton absorption in enhanced green fluorescent protein at the single amino acid residue level.

The chromophore (CRO) of fluorescent proteins (FPs) is embedded in a complex environment that is a source of specific interactions with the CRO. Understanding how these interactions influence FPs spectral properties is important for a directed design of novel markers with desired characteristics. In this work, we apply computational chemistry methods to gain insight into one-, two- and three-photon absorption (1PA, 2PA, 3PA) tuning in enhanced green fluorescent protein (EGFP). To achieve this goal, we built EGFP models differing in: i) number and position of hydrogen-bonds (h-bonds) donors to the CRO and ii) the electric field, as approximated by polarizable force field, acting on the CRO. We find that h-bonding to the CRO's phenolate oxygen results in stronger one- and multiphoton absorption. The brighter absorption can be also achieved by creating more positive electric field near the CRO's phenolate moiety. Interestingly, while individual CRO - environment h-bonds usually enhance 1PA and 2PA, it takes a few h-bond donors to enhance 3PA. Clearly, response of the absorption intensity to many-body effects depends on the excitation mechanism. We further employ symmetry-adapted perturbation theory (SAPT) to reveal excellent (2PA) and good (3PA) correlation of multiphoton intensity with electrostatic and induction interaction energies. This points to importance of accounting for mutual CRO - environment polarization in quantitative calculations of absorption spectra in FPs.

Japanese Encephalitis DNA Vaccines with Epitope Modification Reduce the Induction of Cross-Reactive Antibodies against Dengue Virus and Antibody-Dependent Enhancement of Dengue Virus Infection

Infection with viruses belonging to the genus Flavivirus, such as Japanese encephalitis virus (JEV) and dengue virus (DENV), is a worldwide health problem. Vaccines against JEV and DENV are currently available. However, the dengue vaccine possibly increases the risk of severe dengue due to antibody-dependent enhancement (ADE). Moreover, the Japanese encephalitis (JE) vaccine reportedly induces cross-reactive ADE-prone antibodies against DENV, potentially leading to symptomatic dengue. Therefore, it is necessary to eliminate the risk of ADE through vaccination. In this study, we attempted to develop a JE vaccine that does not induce ADE of DENV infection using an epitope modification strategy. We found that an ADE-prone monoclonal antibody cross-reactive to DENV and JEV recognizes the 106th amino acid residue of the E protein of JEV (E-106). The JE DNA vaccine with a mutation at E-106 (E-106 vaccine) induced comparable neutralizing antibody titers against JEV to those induced by the wild-type JE DNA vaccine. Meanwhile, the E-106 vaccine induced 64-fold less cross-reactive ADE-prone antibodies against DENV. The mutation did not compromise the protective efficacy of the vaccine in the lethal JEV challenge experiment. Altogether, the modification of a single amino acid residue identified in this study helped in the development of an ADE-free JE vaccine.

Active site remodelling of a cyclodipeptide synthase redefines substrate scope

Cyclodipeptide synthases (CDPSs) generate a wide range of cyclic dipeptides using aminoacylated tRNAs as substrates. Histidine-containing cyclic dipeptides have important biological activities as anticancer and neuroprotective molecules. Out of the 120 experimentally validated CDPS members, only two are known to accept histidine as a substrate yielding cyclo(His-Phe) and cyclo(His-Pro) as products. It is not fully understood how CDPSs select their substrates, and we must rely on bioprospecting to find new enzymes and novel bioactive cyclic dipeptides. Here, we developed an in vitro system to generate an extensive library of molecules using canonical and non-canonical amino acids as substrates, expanding the chemical space of histidine-containing cyclic dipeptide analogues. To investigate substrate selection we determined the structure of a cyclo(His-Pro)-producing CDPS. Three consecutive generations harbouring single, double and triple residue substitutions elucidated the histidine selection mechanism. Moreover, substrate selection was redefined, yielding enzyme variants that became capable of utilising phenylalanine and leucine. Our work successfully engineered a CDPS to yield different products, paving the way to direct the promiscuity of these enzymes to produce molecules of our choosing.

Single residue modulators of amyloid formation in the N-terminal P1-region of α-synuclein

Alpha-synuclein (αSyn) is a protein involved in neurodegenerative disorders including Parkinson’s disease. Amyloid formation of αSyn can be modulated by the ‘P1 region’ (residues 36-42). Here, mutational studies of P1 reveal that Y39A and S42A extend the lag-phase of αSyn amyloid formation in vitro and rescue amyloid-associated cytotoxicity in C. elegans. Additionally, L38I αSyn forms amyloid fibrils more rapidly than WT, L38A has no effect, but L38M does not form amyloid fibrils in vitro and protects from proteotoxicity. Swapping the sequence of the two residues that differ in the P1 region of the paralogue γSyn to those of αSyn did not enhance fibril formation for γSyn. Peptide binding experiments using NMR showed that P1 synergises with residues in the NAC and C-terminal regions to initiate aggregation. The remarkable specificity of the interactions that control αSyn amyloid formation, identifies this region as a potential target for therapeutics, despite their weak and transient nature.

Modifying a Hydroxyl Patch in Glucagon-like Peptide 1 Produces Biased Agonists with Unique Signaling Profiles.

Glucagon-like peptide-1 (GLP-1) lowers blood glucose by inducing insulin but also has other poorly understood properties. Here, we show that hydroxy amino acids (Thr11, Ser14, Ser17, Ser18) in GLP-1(7-36) act in concert to direct cell signaling. Mutating any single residue to alanine removes one hydroxyl group, thereby reducing receptor affinity and cAMP 10-fold, with Ala11 or Ala14 also reducing β-arrestin-2 10-fold, while Ala17 or Ala18 also increases ERK1/2 phosphorylation 5-fold. Multiple alanine mutations more profoundly bias signaling, differentially silencing or restoring one or more signaling properties. Mutating three serines silences only ERK1/2, the first example of such bias. Mutating all four residues silences β-arrestin-2, ERK1/2, and Ca2+ maintains the ligand and receptor at the membrane but still potently stimulates cAMP and insulin secretion in cells and mice. These novel findings indicate that hydrogen bonding cooperatively controls cell signaling and highlight an important regulatory hydroxyl patch in hormones that activate class B G protein-coupled receptors.

Control of a far‐red/near‐infrared spectral switch in an artificial fluorescent biliprotein derived from allophycocyanin

The molecular structure of mBDFP, a far-red fluorescent protein (FPs) derived from an allophycocyanin homolog was resolved to 2.52 Å. Its biliverdin chromophore was found to be attached to the protein in an unusual way that was never observed in natural phycobiliproteins, and only once in a sub-population of artificial bacteriophytochrome-derived FPs. One of the biliverdin's vinyl groups had two cysteine residues covalently bound to its two carbon atoms. This reduces the conjugation length of the biliverdin π-electron system, which shifts the absorption and emission spectra by about 40 nm, from the near-infrared to the far-red region of the spectrum. By spectrally characterizing a set of mBDFP mutants, we show that such spectral shifts can be induced by modifying a single residue in either one of two critical positions in the vicinity of the binding cysteines. This changes the reactivity of biliverdin and the cysteine's thiols towards forming one, or two thioether bonds to the vinyl group. The ability to control the spectral properties of BDFP by specific point mutations opens many possibilities for rational design of far-red and near-infrared FPs that are of great interest to the development of fluorescence markers for bioimaging since most biological tissues are transparent in this spectral window.

A single residue confers selective loss of sugar sensing in wrynecks

Sensory receptors evolve, and changes to their response profiles can directly impact sensory perception and affect diverse behaviors, from mate choice to foraging decisions.1-3 Although receptor sensitivities can be highly contingent on changes occurring early in a lineage's evolutionary history,4 subsequent shifts in a species' behavior and ecology may exert selective pressure to modify and even reverse sensory receptor capabilities.5-7 Neither the extent to which sensory reversion occurs nor the mechanisms underlying such shifts is well understood. Using receptor profiling and behavioral tests, we uncover both an early gain and an unexpected subsequent loss of sugar sensing in woodpeckers, a primarily insectivorous family of landbirds.8,9 Our analyses show that, similar to hummingbirds10 and songbirds,4 the ancestors of woodpeckers repurposed their T1R1-T1R3 savory receptor to detect sugars. Importantly, whereas woodpeckers seem to have broadly retained this ability, our experiments demonstrate that wrynecks (an enigmatic ant-eating group sister to all other woodpeckers) selectively lost sugar sensing through a novel mechanism involving a single amino acid change in the T1R3 transmembrane domain. The identification of this molecular microswitch responsible for a sensory shift in taste receptors provides an example ofthe molecular basis of a sensory reversion in vertebrates and offers novel insights into structure-function relationships during sensory receptor evolution.

Identification and quantification of within-burst dynamics in singly labeled single-molecule fluorescence lifetime experiments

Single-molecule spectroscopy has revolutionized molecular biophysics and provided means to probe how structural moieties within biomolecules spatially reorganize at different timescales. There are several single-molecule methodologies that probe local structural dynamics in the vicinity of a single dye-labeled residue, which rely on fluorescence lifetimes as readout. Nevertheless, an analytical framework to quantify dynamics in such single-molecule single dye fluorescence bursts, at timescales of microseconds to milliseconds, has not yet been demonstrated. Here, we suggest an analytical framework for identifying and quantifying within-burst lifetime-based dynamics, such as conformational dynamics recorded in single-molecule photo-isomerization-related fluorescence enhancement. After testing the capabilities of the analysis on simulations, we proceed to exhibit within-burst millisecond local structural dynamics in the unbound α-synuclein monomer. The analytical framework provided in this work paves the way for extracting a full picture of the energy landscape for the coordinate probed by fluorescence lifetime-based single-molecule measurements.

Interdisciplinary in silico studies to understand in-depth molecular level mechanism of drug resistance involving NS3-4A protease of HCV

Hepatitis C virus (HCV) causes hepatitis, a life-threatening disease responsible for liver cirrhosis. Urgent measures have been taken to develop therapeutics against this deadly pathogen. NS3/4A protease is an extremely important target. A series of inhibitors have been developed against this viral protease including Faldaprevir. Unfortunately, the error-prone viral RNA polymerase causes the emergence of resistance, thereby causing reduced effectiveness of those peptidomimetic inhibitors. Among the drug resistant variants, three single amino acid residues (R155, A156 and D168) are notable for their presence in clinical isolates and also their effectivity against most of the known inhibitors in clinical development. Therefore, it is crucial to understand the mechanistic role of those drug resistant variants while designing potent novel inhibitors. In this communication, we have deeply analyzed through using in silico studies to understand the molecular mechanism of alteration of inhibitor binding between wild type and its R155K, A156V and D168V variants. Principal component analysis was carried to identify the backbone fluctuations of important residues in HCV NS3/4A responsible for the inhibitor binding and maintaining drug resistance. Free energy landscape as a function of the principal components has been used to identify the stability and conformation of the key residues that regulate inhibitor binding and their impact in developing drug resistance. Our findings are consistent with the trend of experimental results. The observations are also true in case of other Faldaprevir-like peptidomimetic inhibitors. Understanding this binding mechanism would be significant for the development of novel inhibitors with less susceptibility towards drug resistance. Communicated by Ramaswamy H. Sarma

Discovery of the Key Mutation Site Influencing the Thermostability of Thermomyces lanuginosus Lipase by Rosetta Design Programs.

Lipases are remarkable biocatalysts and are broadly applied in many industry fields because of their versatile catalytic capabilities. Considering the harsh biotechnological treatment of industrial processes, the activities of lipase products are required to be maintained under extreme conditions. In our current study, Gibbs free energy calculations were performed to predict potent thermostable Thermomyces lanuginosus lipase (TLL) variants by Rosetta design programs. The calculating results suggest that engineering on R209 may greatly influence TLL thermostability. Accordingly, ten TLL mutants substituted R209 were generated and verified. We demonstrate that three out of ten mutants (R209H, R209M, and R209I) exhibit increased optimum reaction temperatures, melting temperatures, and thermal tolerances. Based on molecular dynamics simulation analysis, we show that the stable hydrogen bonding interaction between H198 and N247 stabilizes the local configuration of the 250-loop in the three R209 mutants, which may further contribute to higher rigidity and improved enzymatic thermostability. Our study provides novel insights into a single residue, R209, and the 250-loop, which were reported for the first time in modulating the thermostability of TLL. Additionally, the resultant R209 variants generated in this study might be promising candidates for future-industrial applications.