Evidence that the transmembrane domain proximal region of the human T-cell leukemia virus type 1 fusion glycoprotein gp21 has distinct roles in the prefusion and fusion-activated states

To investigate the structural context of the fusion peptide region in human T-cell leukemia virus type 1 gp21, maltose-binding protein (MBP) was used as an N-terminal solubilization partner for the entire gp21 ectodomain (residues 313-445) and C-terminally truncated ectodomain fragments. The bacterial expression of the MBP/gp21 chimeras resulted in soluble trimers containing intramonomer disulfide bonds. Detergents blocked the proteolytic cleavage of fusion peptide residues in the MBP/gp21-(313-425) chimera, indicating that the fusion peptide is available for interaction with detergent despite the presence of an N-terminal MBP domain. Limited proteolysis experiments indicated that the transmembrane domain proximal sequence Thr(425)-Ala(439) protects fusion peptide residues from chymotrypsin. MBP/gp21 chimera stability therefore depends on a functional interaction between N-terminal and transmembrane domain proximal regions in a gp21 helical hairpin structure. In addition, thermal aggregation experiments indicated that the Thr(425)-Ser(436) sequence confers stability to the fusion peptide-containing MBP/gp21 chimeras. The functional role of the transmembrane domain proximal sequence was assessed by alanine-scanning mutagenesis of the full-length envelope glycoprotein, with 11 of 12 single alanine substitutions resulting in 1.5- to 4.5-fold enhancements in cell-cell fusion activity. By contrast, single alanine substitutions in MBP/gp21 did not significantly alter chimera stability, indicating that multiple residues within the transmembrane domain proximal region and the fusion peptide and adjacent glycine-rich segment contribute to stability, thereby mitigating the potential effects of the substitutions. The fusion-enhancing effects of the substitutions are therefore likely to be caused by alteration of the prefusion complex. Our observations suggest that the function of the transmembrane domain proximal sequence in the prefusion envelope glycoprotein is distinct from its role in stabilizing the fusion peptide region in the fusion-activated helical hairpin conformation of gp21.     

Biophysical Characterization of a Vaccine Candidate against HIV-1: The Transmembrane and Membrane Proximal Domains of HIV-1 gp41 as a Maltose Binding Protein Fusion

The membrane proximal region (MPR, residues 649–683) and transmembrane domain (TMD, residues 684–705) of the gp41 subunit of HIV-1’s envelope protein are highly conserved and are important in viral mucosal transmission, virus attachment and membrane fusion with target cells. Several structures of the trimeric membrane proximal external region (residues 662–683) of MPR have been reported at the atomic level; however, the atomic structure of the TMD still remains unknown. To elucidate the structure of both MPR and TMD, we expressed the region spanning both domains, MPR-TM (residues 649–705), in Escherichia coli as a fusion protein with maltose binding protein (MBP). MPR-TM was initially fused to the C-terminus of MBP via a 42 aa-long linker containing a TEV protease recognition site (MBP-linker-MPR-TM). Biophysical characterization indicated that the purified MBP-linker-MPR-TM protein was a monodisperse and stable candidate for crystallization. However, crystals of the MBP-linker-MPR-TM protein could not be obtained in extensive crystallization screens. It is possible that the 42 residue-long linker between MBP and MPR-TM was interfering with crystal formation. To test this hypothesis, the 42 residue-long linker was replaced with three alanine residues. The fusion protein, MBP-AAA-MPR-TM, was similarly purified and characterized. Significantly, both the MBP-linker-MPR-TM and MBP-AAA-MPR-TM proteins strongly interacted with broadly neutralizing monoclonal antibodies 2F5 and 4E10. With epitopes accessible to the broadly neutralizing antibodies, these MBP/MPR-TM recombinant proteins may be in immunologically relevant conformations that mimic a pre-hairpin intermediate of gp41.     

The extracellular domain of immunodeficiency virus gp41 protein: expression in Escherichia coli, purification, and crystallization.

The env gene of SIV and HIV-1 encodes a single glycoprotein gp 160, which is processed to give a noncovalent complex of the soluble glycoprotein gp120 and the transmembrane glycoprotein gp41. The extracellular region (ectodomain), minus the N-terminal fusion peptide, of gp41 from HIV-1 (residues 27-154) and SIV (residues 27-149) have been expressed in Escherichia coli. These insoluble proteins were solubilized and subjected to a simple purification and folding scheme, which results in high yields of soluble protein. Purified proteins have a trimeric subunit composition and high alpha-helical content, consistent with the predicted coil-coil structure. SIV gp41 containing a double cysteine mutation was crystallized. The crystals are suitable for X-ray structure determination and, preliminary analysis, together with additional biochemical evidence, indicates that the gp41 trimer is arranged as a parallel bundle with threefold symmetry.     

Identification of a conserved domain of the HIV-1 transmembrane protein gp41 which interacts with cholesteryl groups

A soluble form of the HIV-1 envelope glycoprotein gp160 devoid of the transmembrane anchor domain was found to bind to cholesteryl-hemisuccinate agarose. The external subunit gp120 failed to bind to the resin, suggesting that the site responsible for the binding to cholesterol was located in the transmembrane protein gp41. We constructed a series of maltose binding protein (MBP) fusion proteins representing overlapping fragments of the gp41 molecule and we studied their capacity to bind to cholesteryl beads. The domain responsible for binding to cholesterol was localised within the residues 668 to 684 immediately adjacent to the membrane spanning domain. We identified a short sequence (LWYIK, aa 678-683) comparable to the cholesterol interaction amino acid consensus pattern published by Li and Papadopoulos [Endocrinology 139 (1998) 4991]. We demonstrated that the sequence LWYIK synthesized fused to the MBP was able to bind to cholesteryl groups. A synthetic peptide containing the sequence LWYIK was found to inhibit the interaction between cholesteryl beads and MBP44, an MBP fusion HIV-1 envelope protein that contains the putative cholesterol binding domain. Human sera obtained from HIV-1 seropositive patients did not react in ELISA to the LWYIK sequence, suggesting that this region is not exposed to the immune system. The biological significance of the interaction between gp41 and cholesterol is discussed.     

Expression and biochemical analysis of the entire HIV-2 gp41 ectodomain: determinants of stability map to N- and C-terminal sequences outside the 6-helix bundle core

The folding of HIV gp41 into a 6-helix bundle drives virus-cell membrane fusion. To examine the structural relationship between the 6-helix bundle core domain and other regions of gp41, we expressed in Escherichia coli, the entire ectodomain of HIV-2(ST) gp41 as a soluble, trimeric maltose-binding protein (MBP)/gp41 chimera. Limiting proteolysis indicated that the Cys-591-Cys-597 disulfide-bonded region is outside a core domain comprising two peptides, Thr-529-Trp-589 and Val-604-Ser-666. A biochemical examination of MBP/gp41 chimeras encompassing these core peptides indicated that the N-terminal polar segment, 521-528, and C-terminal membrane-proximal segment, 658-666, cooperate in stabilizing the ectodomain. A functional interaction between sequences outside the gp41 core may contribute energy to membrane fusion.     

Virus Membrane Fusion Proteins: Biological Machines that Undergo a Metamorphosis

Fusion proteins from a group of widely disparate viruses, including the paramyxovirus F protein, the HIV and SIV gp160 proteins, the retroviral Env protein, the Ebola virus Gp, and the influenza virus haemagglutinin, share a number of common features. All contain multiple glycosylation sites, and must be trimeric and undergo proteolytic cleavage to be fusogenically active. Subsequent to proteolytic cleavage, the subunit containing the transmembrane domain in each case has an extremely hydrophobic region, termed the fusion peptide, or at near its newly generated N-terminus. In addition, all of these viral fusion proteins have 4–3 heptad repeat sequences near both the fusion peptide and the transmembrane domain. These regions have been demonstrated from a tight complex, in which the N-terminal heptad repeat forms a trimeric-coiled coil, with the C-terminal heptad repeat forming helical regions that buttress the coiled-coil in an anti-parallel manner. The significance of each of these structuralelements in the processing and function of these viral fusion proteins is discussed.     

杜氏盐藻核基质结合区结合蛋白的细胞定位

为研究核基质结合区结合蛋白的功能及调控机制,PCR扩增杜氏盐藻MBP的cDNA全长序列及N端和C端序列,与绿色荧光蛋白基因融合构建真核表达载体,脂质体转染CHO细胞,Western blotting和荧光显微镜检测基因表达情况和细胞定位。结果显示:MBP及N端和C端融合蛋白成功在CHO细胞表达,MBP和C端部分定位于细胞核且聚集于核仁,N端部分分布整个细胞,说明MBP定位于细胞核且细胞定位信号位于C端,MBP可能与rRNA前体结合发挥作用。     

Crystal structures of MBP fusion proteins

Although chaperone‐assisted protein crystallization remains a comparatively rare undertaking, the number of crystal structures of polypeptides fused to maltose‐binding protein (MBP) that have been deposited in the Protein Data Bank (PDB) has grown dramatically during the past decade. Altogether, 102 fusion protein structures were detected by Basic Local Alignment Search Tool (BLAST) analysis. Collectively, these structures comprise a range of sizes, space groups, and resolutions that are typical of the PDB as a whole. While most of these MBP fusion proteins were equipped with short inter‐domain linkers to increase their rigidity, fusion proteins with long linkers have also been crystallized. In some cases, surface entropy reduction mutations in MBP appear to have facilitated the formation of crystals. A comparison of the structures of fused and unfused proteins, where both are available, reveals that MBP‐mediated structural distortions are very rare.     

Design and properties of N(CCG)-gp41, a chimeric gp41 molecule with nanomolar HIV fusion inhibitory activity.

The design and characterization of a chimeric protein, termed N(CCG)-gp41, derived from the ectodomain of human immunodeficiency virus (HIV), type I gp41 is described. N(CCG)-gp41 features an exposed trimeric coiled-coil comprising the N-terminal helices of the gp41 ectodomain. The trimeric coiled-coil is stabilized both by fusion to a minimal thermostable ectodomain of gp41 and by engineered intersubunit disulfide bonds. N(CCG)-gp41 is shown to inhibit HIV envelope-mediated cell fusion at nanomolar concentrations with an IC(50) of 16.1 +/- 2.8 nm. It is proposed that N(CCG)-gp41 targets the exposed C-terminal region of the gp41 ectodomain in its pre-hairpin intermediate state, thereby preventing the formation of the fusogenic form of the gp41 ectodomain, which comprises a highly stable trimer of hairpins arranged in a six-helix bundle. N(CCG)-gp41 has potential as a therapeutic agent for the direct inhibition of HIV cell entry, as an anti-HIV vaccine, and as a component of a rapid throughput assay for screening for small molecule inhibitors of HIV envelope-mediated cell fusion. It is anticipated that antibodies raised against N(CCG)-gp41 may target the trimeric coiled-coil of N-terminal helices of the gp41 ectodomain that is exposed in the pre-hairpin intermediate state in a manner analogous to peptides derived from the C-terminal helix of gp41 that are currently in clinical trials.     

Conformation of trimeric envelope glycoproteins: the CD4-dependent membrane fusion mechanism of HIV-1

The HIV-1 envelope glycoproteins are assembled by the trimeric gp120s and gp41s proteins. The gp120 binds sequentially to CD4 and coreceptor for initiating virus entry. Because of noncovalent interaction and heavy glycosylation for envelope glycoproteins, it is highly difficult to determine entire envelope glycoproteins structure now. Such question extremely limits our good understanding of HIV-1 membrane fusion mechanism. Here, a novel and reasonable assembly model of trimeric gp120s and gp41s was proposed based on the conformational dynamics of trimeric gp120-gp41 complex and gp41, respectively. As for gp41, the heptad repeat sequences in the gp41 C-terminal is of enormous flexibility. On the contrary, the heptad repeat sequences in the gp41 N-terminal likely present stable three-helical bundle due to strong nonpolar interaction, and they were predicted to associate three alpha1 helixes from the non-neutralizing face of the gp120 inner domain, which is quite similar to gp41 fusion core structure. Such interaction likely leads to the formation of noncovalent gp120-gp41 complex. In the proposed assembly of trimeric gp120-gp41 complex, three gp120s present not only perfectly complementary and symmetrical distribution around the gp41, but also different flexibility degree in the different structural domains. Thus, the new model can well explain numerous experimental phenomena, present plenty of structural information, elucidate effectively HIV-1 membrane fusion mechanism, and direct to further develop vaccine and novel fusion inhibitors.     

Functional Analysis of the Disulfide-Bonded Loop/Chain Reversal Region of Human Immunodeficiency Virus Type 1 gp41 Reveals a Critical Role in gp120-gp41 Association

Human immunodeficiency virus type 1 (HIV-1) entry into cells is mediated by the surface-exposed envelope protein (SU) gp120, which binds to cellular CD4 and chemokine receptors, triggering the membrane fusion activity of the transmembrane (TM) protein gp41. The core of gp41 comprises an N-terminal triple-stranded coiled coil and an antiparallel C-terminal helical segment which is packed against the exterior of the coiled coil and is thought to correspond to a fusion-activated conformation. The available gp41 crystal structures lack the conserved disulfide-bonded loop region which, in human T-lymphotropic virus type 1 (HTLV-1) and murine leukemia virus TM proteins, mediates a chain reversal, connecting the antiparallel N- and C-terminal regions. Mutations in the HTLV-1 TM protein gp21 disulfide-bonded loop/chain reversal region adversely affected fusion activity without abolishing SU-TM association (A. L. Maerz, R. J. Center, B. E. Kemp, B. Kobe, and P. Poumbourios, J. Virol. 74:6614-6621, 2000). We now report that in contrast to our findings with HTLV-1, conservative substitutions in the HIV-1 gp41 disulfide-bonded loop/chain reversal region abolished association with gp120. While the mutations affecting gp120-gp41 association also affected cell-cell fusion activity, HIV-1 glycoprotein maturation appeared normal. The mutant glycoproteins were processed, expressed at the cell surface, and efficiently immunoprecipitated by conformation-dependent monoclonal antibodies. The gp120 association site includes aromatic and hydrophobic residues on either side of the gp41 disulfide-bonded loop and a basic residue within the loop. The HIV-1 gp41 disulfide-bonded loop/chain reversal region is a critical gp120 contact site; therefore, it is also likely to play a central role in fusion activation by linking CD4 plus chemokine receptor-induced conformational changes in gp120 to gp41 fusogenicity. These gp120 contact residues are present in diverse primate lentiviruses, suggesting conservation of function.     

Crystal structures of fusion proteins with large-affinity tags

The fusion of a protein of interest to a large-affinity tag, such as the maltose-binding protein (MBP), thioredoxin (TRX), or glutathione-S-transferase (GST), can be advantageous in terms of increased expression, enhanced solubility, protection from proteolysis, improved folding, and protein purification via affinity chromatography. Unfortunately, crystal growth is hindered by the conformational heterogeneity induced by the fusion tag, requiring that the tag is removed by a potentially problematic cleavage step. The first three crystal structures of fusion proteins with large-affinity tags have been reported recently. All three structures used a novel strategy to rigidly fuse the protein of interest to MBP via a short three- to five-amino acid spacer. This strategy has the potential to aid structure determination of proteins that present particular experimental challenges and are not conducive to more conventional crystallization strategies (e.g., membrane proteins). Structural genomics initiatives may also benefit from this approach as a way to crystallize problematic proteins of significant interest.     

Highly stable trimers formed by human immunodeficiency virus type 1 envelope glycoproteins fused with the trimeric motif of T4 bacteriophage fibritin

The envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1) function as a trimer composed of three gp120 exterior glycoproteins and three gp41 transmembrane proteins. Soluble gp140 glycoproteins composed of the uncleaved ectodomains of gp120 and gp41 form unstable, heterogeneous oligomers, but soluble gp140 trimers can be stabilized by fusion with a C-terminal, trimeric GCN4 motif (X. Yang et al., J. Virol. 74:5716-5725, 2000). To understand the influence of the C-terminal trimerization domain on the properties of soluble HIV-1 envelope glycoprotein trimers, uncleaved, soluble gp140 glycoproteins were stabilized by fusion with another trimeric motif derived from T4 bacteriophage fibritin. The fibritin construct was more stable to heat and reducing conditions than the GCN4 construct. Both GCN4- and fibritin-stabilized soluble gp140 glycoproteins exhibited patterns of neutralizing and nonneutralizing antibody binding expected for the functional envelope glycoprotein spike. Of note, two potently neutralizing antibodies, immunoglobulin G1b12 and 2G12, exhibited the greatest recognition of the stabilized, soluble trimers, relative to recognition of the gp120 monomer. The observed similarities between the GCN4 and fibritin constructs indicate that the HIV-1 envelope glycoprotein ectodomains dictate many of the antigenic and structural features of these fusion proteins. The melting temperatures and ligand recognition properties of the GCN4- and fibritin-stabilized soluble gp140 glycoproteins suggest that these molecules assume conformations distinct from that of the fusion-active, six-helix bundle.     

Construction, expression, and analysis of recombinant HIV gp41 constructs containing a novel cellular binding domain

The gp41 polypeptide of human immunodeficiency virus (HIV) contains an immunosuppressive domain, an epitope which elicits specific cytolytic T cell responses to HIV, and a complement Clq interactive domain. In addition, a synthetic peptide called CS3, derived from gp41 (amino acids 576-593 of gp160) and contiguous with the major immunodominant domain, binds to cellular proteins and may be important in HIV entry/fusion. In order to further investigate the role of the CS3 region of gp41 in cellular binding and to investigate other properties of gp41, sufficient quantities of this polypeptide must be readily available. We have therefore cloned the region of the HIV genome between nucleotides 7891 and 8188 (corresponding to amino acids 541-639 of gp160) into a series of procaryotic expression vectors. The resulting clones express a recombinant polypeptide of gp41 (r41). Two of these recombinants, pMAL-cRl/r41 and pGEMEX-2/r41, expressed the highest and most consistent levels of r41 as judged by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. With the pMAL-cRl/r41 construct, r41 was expressed as a fusion to the maltose-binding protein (MBP) and, following purification by affinity chromatography, was cleaved from MBP by factor Xa protease digestion. MBP/r41 may be useful for studies of a reported gp41 cellular binding domain and may facilitate studies involving other functions ascribed to this region of gp41. (c) 1993 John Wiley & Sons, Inc.     

Design of a novel peptide inhibitor of HIV fusion that disrupts the internal trimeric coiled-coil of gp41

The pre-hairpin intermediate of gp41 from the human immunodeficiency virus (HIV) is the target for two classes of fusion inhibitors that bind to the C-terminal region or the trimeric coiled-coil of N-terminal helices, thereby preventing formation of the fusogenic trimer of hairpins. Using rational design, two 36-residue peptides, N36(Mut(e,g)) and N36(Mut(a,d)), were derived from the parent N36 peptide comprising the N-terminal helix of the gp41 ectodomain (residues 546-581 of HIV-1 envelope), characterized by analytical ultracentrifugation and CD, and assessed for their ability to inhibit HIV fusion using a quantitative vaccinia virus-based fusion assay. N36(Mut(e,g)) contains nine amino acid substitutions designed to disrupt interactions with the C-terminal region of gp41 while preserving contacts governing the formation of the trimeric coiled-coil. N36(Mut(a,d)) contains nine substitutions designed to block formation of the trimeric coiled-coil but retains residues that interact with the C-terminal region of gp41. N36(Mut(a,d)) is monomeric, is largely random coil, does not interact with the C34 peptide derived from the C-terminal region of gp41 (residues 628-661), and does not inhibit fusion. The trimeric coiled-coil structure is therefore a prerequisite for interaction with the C-terminal region of gp41. N36(Mut(e,g)) forms a monodisperse, helical trimer in solution, does not interact with C34, and yet inhibits fusion about 50-fold more effectively than the parent N36 peptide (IC(50) approximately 308 nm versus approximately 16 microm). These results indicate that N36(Mut(e,g)) acts by disrupting the homotrimeric coiled-coil of N-terminal helices in the pre-hairpin intermediate to form heterotrimers. Thus N36(Mut(e,g)) represents a novel third class of gp41-targeted HIV fusion inhibitor. A quantitative model describing the interaction of N36(Mut(e,g)) with the pre-hairpin intermediate is presented.     

On the interaction between gp41 and membranes: the immunodominant loop stabilizes gp41 helical hairpin conformation

gp41 is the protein responsible for the process of membrane fusion that allows primate lentiviruses (HIV and SIV) to enter into their host cells. gp41 ectodomain contains an N-terminal and a C-terminal heptad repeat region (NHR and CHR) connected by an immunodominant loop. In the absence of membranes, the NHR and CHR segments fold into a protease-resistant core with a trimeric helical hairpin structure. However, when the immunodominant loop is not present (either in a complex formed by HIV-1 gp41-derived NHR and CHR peptides or by mild treatment with protease of recombinant constructs of HIV-1 gp41 ectodomain, which also lack the N-terminal fusion peptide and the C-terminal Trp-rich region) membrane binding induces a conformational change in the gp41 core structure. Here, we further investigated whether covalently linking the NHR and CHR segments by the immunodominant loop affects this conformational change. Specifically, we analyzed a construct corresponding to a fragment of SIVmac239 gp41ectodomain (residues 27-149, named e-gp41) by means of surface plasmon resonance, Trp and rhodamine fluorescence, ATR-FTIR spectroscopy, and differential scanning calorimetry. Our results suggest that the presence of the loop stabilizes the trimeric helical hairpin both when e-gp41 is in aqueous solution and when it is bound to the membrane surface. Bearing in mind possible differences between HIV-1 and SIV gp41, and considering that the gp41 ectodomain constructs analyzed to date lack the N-terminal fusion peptide and the C-terminal Trp-rich region, we discuss our observations in relation to the mechanism of virus-induced membrane fusion.     

Bacteriophage P22 tail accessory factor GP26 is a long triple-stranded coiled-coil

P22 is a well characterized tailed bacteriophage that infects Salmonella enterica serovar Typhimurium. It is characterized by a "short" tail, which is formed by five proteins: the dodecameric portal protein (gp1), three tail accessory factors (gp4, gp10, gp26), and six trimeric copies of the tail-spike protein (gp9). We have isolated the gene encoding tail accessory factor gp26, which is responsible for stabilization of viral DNA within the mature phage, and using a variety of biochemical and biophysical techniques we show that gp26 is very likely a triple stranded coiled-coil protein. Electron microscopic examination of purified gp26 indicates that the protein adopts a rod-like structure approximately 210 angstroms in length. This trimeric rod displays an exceedingly high intrinsic thermostability (T(m) approximately 85 degrees C), which suggests a potentially important structural role within the phage tail apparatus. We propose that gp26 forms the thin needle-like fiber emanating from the base of the P22 neck that has been observed by electron microscopy of negatively stained P22 virions. By analogy with viral trimeric coiled-coil class I membrane fusion proteins, gp26 may represent the membrane-penetrating device used by the phage to pierce the host outer membrane.     

Synthesis,enhanced fusogenicity,and solid state NMR measurements of cross-linked HIV-1 fusion peptides

In the HIV-1 gp41 and other viral fusion proteins, the minimal oligomerization state is believed to be trimeric with three N-terminal fusion peptides inserting into the membrane in close proximity. Previous studies have demonstrated that the fusion peptide by itself serves as a useful model fusion system, at least to the hemifusion stage in which the viral and target cell lipids are mixed. In the present study, HIV-1 fusion peptides were chemically synthesized and cross-linked at their C-termini to form dimers or trimers. C-terminal trimerization is their likely topology in the fusogenic form of the intact gp41 protein. The fusogenicity of the peptides was then measured in an intervesicle lipid mixing assay, and the assay results were compared to those of the monomer. For monomer, dimer, and trimer at peptide strand/lipid mol ratios between 0.0050 and 0.010, the final extent of lipid mixing for the dimer and trimer was 2-3 times greater than for the monomer. These data suggest that the higher local concentration of peptide strands in the cross-linked peptides enhances fusogenicity and that oligomerization of the fusion peptide in gp41 may enhance the rate of viral/target cell membrane fusion. For gp41, this effect is in addition to the role of the trimeric coiled-coil structure in bringing about apposition of viral and target cell membranes. NMR measurements on the membrane-associated dimeric fusion peptide were consistent with an extended structure at Phe-8, which is the same as has been observed for the membrane-bound monomer in the same lipid composition.     

A synergistic approach to protein crystallization: Combination of a fixed‐arm carrier with surface entropy reduction

Protein crystallographers are often confronted with recalcitrant proteins not readily crystallizable, or which crystallize in problematic forms. A variety of techniques have been used to surmount such obstacles: crystallization using carrier proteins or antibody complexes, chemical modification, surface entropy reduction, proteolytic digestion, and additive screening. Here we present a synergistic approach for successful crystallization of proteins that do not form diffraction quality crystals using conventional methods. This approach combines favorable aspects of carrier‐driven crystallization with surface entropy reduction. We have generated a series of maltose binding protein (MBP) fusion constructs containing different surface mutations designed to reduce surface entropy and encourage crystal lattice formation. The MBP advantageously increases protein expression and solubility, and provides a streamlined purification protocol. Using this technique, we have successfully solved the structures of three unrelated proteins that were previously unattainable. This crystallization technique represents a valuable rescue strategy for protein structure solution when conventional methods fail.