A Drosophila model of ALS: human ALS-associated mutation in VAP33A suggests a dominant negative mechanism

ALS8 is caused by a dominant mutation in an evolutionarily conserved protein, VAPB (vesicle-associated membrane protein (VAMP)-associated membrane protein B)/ALS8). We have established a fly model of ALS8 using the corresponding mutation in Drosophila VAPB (dVAP33A) and examined the effects of this mutation on VAP function using genetic and morphological analyses. By simultaneously assessing the effects of VAP(wt) and VAP(P58S) on synaptic morphology and structure, we demonstrate that the phenotypes produced by neuronal expression of VAP(P58S) resemble VAP loss of function mutants and are opposite those of VAP overexpression, suggesting that VAP(P58S) may function as a dominant negative. This is brought about by aggregation of VAP(P58S) and recruitment of wild type VAP into these aggregates. Importantly, we also demonstrate that the ALS8 mutation in dVAP33A interferes with BMP signaling pathways at the neuromuscular junction, identifying a new mechanism underlying pathogenesis of ALS8. Furthermore, we show that mutant dVAP33A can serve as a powerful tool to identify genetic modifiers of VAPB. This new fly model of ALS, with its robust pathological phenotypes, should for the first time allow the power of unbiased screens in Drosophila to be applied to study of motor neuron diseases.     

A highly conserved binding site in vesicle-associated membrane protein-associated protein (VAP) for the FFAT motif of lipid-binding proteins

A variety of lipid-binding proteins contain a recently described motif, designated FFAT (two phenylalanines in an acidic tract), which binds to vesicle-associated-membrane protein-associated protein (VAP). VAP is a conserved integral membrane protein of the endoplasmic reticulum that contains at its amino terminus a domain related to the major sperm protein of nematode worms. Here we have studied the FFAT-VAP interaction in Saccharomyces cerevisiae, where the VAP homologue Scs2 regulates phospholipid metabolism via an interaction with the FFAT motif of Opi1. By introducing mutations at random into Scs2, we found that mutations that abrogated binding to FFAT were clustered in the most highly conserved region. Using site-directed mutagenesis, we identified several critical residues, including two lysines widely separated in the primary sequence. By examining all other conserved basic residues, we identified a third residue that was moderately important for binding FFAT. Modeling VAP on the known structure of major sperm protein showed that the critical residues form a patch on a positively charged face of the protein. In vivo functional studies of SCS22, a second SCS2-like gene in S. cerevisiae, showed that SCS2 was the dominant gene in the regulation of Opi1, with a minor contribution from SCS22. We then established that reduction in the affinity of Scs2 mutants for FFAT correlated well with loss of function, indicating the importance of these residues for binding FFAT motifs. Finally, we found that human VAP-A could substitute for Scs2 but that it functioned poorly, suggesting that other factors modulate the binding of Scs2 to proteins with FFAT motifs.     

The ALS8 protein VAPB interacts with the ER–Golgi recycling protein YIF1A and regulates membrane delivery into dendrites

The vesicle‐associated membrane protein (VAMP) associated protein B (VAPB) is an integral membrane protein localized to the endoplasmic reticulum (ER). The P56S mutation in VAPB has been linked to motor neuron degeneration in amyotrophic lateral sclerosis type 8 (ALS8) and forms ER‐like inclusions in various model systems. However, the role of wild‐type and mutant VAPB in neurons is poorly understood. Here, we identified Yip1‐interacting factor homologue A (YIF1A) as a new VAPB binding partner and important component in the early secretory pathway. YIF1A interacts with VAPB via its transmembrane regions, recycles between the ER and Golgi and is mainly localized to the ER–Golgi intermediate compartments (ERGICs) in rat hippocampal neurons. VAPB strongly affects the distribution of YIF1A and is required for intracellular membrane trafficking into dendrites and normal dendritic morphology. When VAPB‐P56S is present, YIF1A is recruited to the VAPB‐P56S clusters and loses its ERGIC localization. These data suggest that both VAPB and YIF1A are important for ER‐to‐Golgi transport and that missorting of YIF1A may contribute to VAPB‐associated motor neuron disease.     

Role of conserved prolines in the structure and function of the Na+/dicarboxylate cotransporter 1, NaDC1

The Na+/dicarboxylate cotransporter 1 (NaDC1) is a low-affinity transporter for citric acid cycle intermediates such as succinate and citrate. The sequence of NaDC1 contains a number of conserved proline residues in predicted transmembrane helices (TMs) 7 and 10. These transmembrane domains are of particular importance because they may be involved in determining the substrate or cation-binding affinity in NaDC1. Four conserved proline residues in TMs 7 and 10 of rabbit NaDC1 were replaced with alanine to promote ideal alpha helix or glycine to promote free conformation, and the mutant transporters were expressed in the HRPE cell line. Mutations of prolines in TM 10 produced decreased protein expression and activity, whereas mutations of prolines in TM 7 completely abolished protein expression and activity. The chemical chaperone glycerol was found to improve the expression of the Pro-351 mutants in TM 7, suggesting that these mutants had defects in trafficking. The inactive mutant transporters at position 351 could also be rescued by the addition of a proline at a second site. For example, the P351A-F347P mutant had restored activity, although its substrate specificity was altered. We conclude that, in TM 7, Pro-327 may be of particular importance in the function of the transporter, whereas Pro-351 may affect protein targeting. The prolines in TM 10, at positions 523 and 524, may not be directly involved in the transporter function but may be necessary for maintaining structure.     

The yeast VAP homolog Scs2p has a phosphoinositide-binding ability that is correlated with its activity

The yeast VAMP-associated protein (VAP) homolog Scs2p is an endoplasmic reticulum (ER)/nuclear membrane protein that binds to an FFAT (diphenylalanine in an acidic tract) motif found in various lipid-metabolic proteins, including Opi1p, a negative regulator of phospholipid biosynthesis. Here, we show that Scs2p is a novel phosphoinositide-binding protein that can bind to phosphatidylinositol monophosphates and bisphosphates in vitro. The phosphoinositide-binding domain was assigned to the N-terminal major sperm protein (MSP) domain which also contains the FFAT-binding domain. When several lysine residues in the MSP domain were substituted for alanine, the resulting mutant Scs2 proteins lost the phosphoinositide-binding ability and failed to complement the inositol auxotrophy of an scs2 deletion strain. However, the mutant proteins still localized in the ER/nuclear membrane, in a similar manner to wild-type Scs2p. These results suggest the possibility that Scs2p activity is regulated by phosphoinositides to coordinate phospholipid biosynthesis in response to changes in phospholipid composition.     

The ALS8-associated mutant VAPB(P56S) is resistant to proteolysis in neurons

VAMP/synaptobrevin associated proteins A and B (VAPA and VAPB), are type IV membrane proteins enriched on ER and Golgi membranes. Both VAPA and B interact with cytoplasmic lipid transport proteins and cytoskeletal elements to maintain the structure and composition of ER and Golgi membranes. Truncated forms of both proteins are present in some tissues but the functional significance of this is not clear. In rodents processing of VAPA occurs in most tissues, however, truncated forms of VAPB have only been reported in brain tissue. It is demonstrated here that the extent of VAPB processing in rat increases during postnatal development and that it is restricted to neurons. The C-terminal polypeptide generated by this cleavage reaction remains associated with cell membranes, but its subcellular distribution is distinct from the full-length protein. A mutant form of VAPB is associated with a familial form of neurodegenerative disease, amyotrophic lateral sclerosis type 8. The mutant protein, VAPB(P56S) , is resistant to truncation in primary neuronal cultures, although remains sensitive to some form of proteolysis when over-expressed in HEK293 cells. These data suggest that neuronal cells have a particular requirement for VAPB proteolysis and that reduced levels of processed polypeptides may contribute to the neurodegeneration associated with amyotrophic lateral sclerosis type 8.     

Characterization of amyotrophic lateral sclerosis-linked P56S mutation of vesicle-associated membrane protein-associated protein B (VAPB/ALS8)

The P56S mutation in VAPB (vesicle-associated membrane protein-associated protein B) causes autosomal dominant motoneuronal diseases. Although it was reported that the P56S mutation induces localization shift of VAPB from endoplasmic reticulum (ER) to non-ER compartments, it remains unclear what the physiological function of VAPB is and how the P56S mutation in VAPB causes motoneuronal diseases. Here we demonstrate that overexpression of wild type VAPB (wt-VAPB) promotes unfolded protein response (UPR), which is an ER reaction to suppress accumulation of misfolded proteins, and that small interfering RNA for VAPB attenuates UPR to chemically induced ER stresses, indicating that VAPB is physiologically involved in UPR. The P56S mutation nullifies the function of VAPB to mediate UPR by inhibiting folding of VAPB that results in insolubility and aggregate formation of VAPB in non-ER fractions. Furthermore, we have found that expression of P56S-VAPB inhibits UPR, mediated by endogenous wt-VAPB, by inducing aggregate formation and mislocalization into non-ER fractions of wt-VAPB. Consequently, the P56S mutation in a single allele of the VAPB gene may diminish the activity of VAPB to mediate UPR to less than half the normal level. We thus speculate that the malfunction of VAPB to mediate UPR, caused by the P56S mutation, may contribute to the development of motoneuronal degeneration linked to VAPB/ALS8.     

The amyotrophic lateral sclerosis 8 protein VAPB is cleaved, secreted, and acts as a ligand for Eph receptors

VAP proteins (human VAPB/ALS8, Drosophila VAP33, and C. elegans VPR-1) are homologous proteins with an amino-terminal major sperm protein (MSP) domain and a transmembrane domain. The MSP domain is named for its similarity to the C. elegans MSP protein, a sperm-derived hormone that binds to the Eph receptor and induces oocyte maturation. A point mutation (P56S) in the MSP domain of human VAPB is associated with Amyotrophic lateral sclerosis (ALS), but the mechanisms underlying the pathogenesis are poorly understood. Here we show that the MSP domains of VAP proteins are cleaved and secreted ligands for Eph receptors. The P58S mutation in VAP33 leads to a failure to secrete the MSP domain as well as ubiquitination, accumulation of inclusions in the endoplasmic reticulum, and an unfolded protein response. We propose that VAP MSP domains are secreted and act as diffusible hormones for Eph receptors. This work provides insight into mechanisms that may impact the pathogenesis of ALS.     

Perturbation of Critical Prolines in Gloeobacter violaceus Ligand-gated Ion Channel (GLIC) Supports Conserved Gating Motions among Cys-loop Receptors

Gloeobacter violaceus ligand-gated ion channel (GLIC) has served as a valuable structural and functional model for the eukaryotic Cys-loop receptor superfamily. In Cys-loop and other receptors, we have previously demonstrated the crucial roles played by several conserved prolines. Here we explore the role of prolines in the gating transitions of GLIC. As conventional substitutions at some positions resulted in nonfunctional proteins, we used in vivo non-canonical amino acid mutagenesis to determine the specific structural requirements at these sites. Receptors were expressed heterologously in Xenopus laevis oocytes, and whole-cell electrophysiology was used to monitor channel activity. Pro-119 in the Cys-loop, Pro-198 and Pro-203 in the M1 helix, and Pro-299 in the M4 helix were sensitive to substitution, and distinct roles in receptor activity were revealed for each. In the context of the available structural data for GLIC, the behaviors of Pro-119, Pro-203, and Pro-299 mutants are consistent with earlier proline mutagenesis work. However, the Pro-198 site displays a unique phenotype that gives evidence of the importance of the region surrounding this residue for the correct functioning of GLIC.     

Prion Protein Prolines 102 and 105 and the Surrounding Lysine Cluster Impede Amyloid Formation

Human prion diseases can have acquired, sporadic, or genetic origins, each of which results in the conversion of prion protein (PrP) to transmissible, pathological forms. The genetic prion disease Gerstmann-Straussler-Scheinker syndrome can arise from point mutations of prolines 102 or 105. However, the structural effects of these two prolines, and mutations thereof, on PrP misfolding are not well understood. Here, we provide evidence that individual mutations of Pro-102 or Pro-105 to noncyclic aliphatic residues such as the Gerstmann-Straussler-Scheinker-linked leucines can promote the in vitro formation of PrP amyloid with extended protease-resistant cores reminiscent of infectious prions. This effect was enhanced by additional charge-neutralizing mutations of four nearby lysine residues comprising the so-called central lysine cluster. Substitution of these proline and lysine residues accelerated PrP conversion such that spontaneous amyloid formation was no longer slower than scrapie-seeded amyloid formation. Thus, Pro-102 and Pro-105, as well as the lysines in the central lysine cluster, impede amyloid formation by PrP, implicating these residues as key structural modulators in the conversion of PrP to disease-associated types of amyloid.     

First evidence of pathogenicity of V234I mutation of hVAPB found in Amyotrophic Lateral Sclerosis

Amyotrophic Lateral Sclerosis is a motor neurodegenerative disease which is characterized by progressive loss of motor neurons followed by paralysis and eventually death. In human, VAMP-associated protein B (VAPB) is the causative gene of the familial form of ALS8. Previous studies have shown that P56S and T46I point mutations of hVAPB are present in this form of ALS. Recently, another mutation, V234I of hVAPB was found in one familial case of ALS. This is the first study where we have shown that V234I-VAPB does not form aggregate like other two mutants of VAPB and localizes differently than the wild type VAPB. It induces Ubiquitin aggregation followed by cell death. We propose that V234I-VAPB exhibits the characteristics of ALS in spite of not having the typical aggregation property of different mutations in various neurodegenerative diseases.     

CYP2E1 and Parkinson’s disease in a MPTP-induced C57BL/6 mouse model

Background

A proline-to-serine substitution at position-56 (P56S) of vesicle-associated membrane protein-associated protein B (VAPB) causes a form of dominantly inherited motor neuron disease (MND), including typical and atypical amyotrophic lateral sclerosis (ALS) and a mild late-onset spinal muscular atrophy (SMA). VAPB is an integral endoplasmic reticulum (ER) protein and has been implicated in various cellular processes, including ER stress, the unfolded protein response (UPR) and Ca²⁺ homeostasis. However, it is unclear how the P56S mutation leads to neurodegeneration and muscle atrophy in patients. The formation of abnormal VAPB-positive inclusions by mutant VAPB suggests a possible toxic gain of function as an underlying mechanism. Furthermore, the amount of VAPB protein is reported to be reduced in sporadic ALS patients and mutant SOD1G93A mice, leading to the hypothesis that wild type VAPB plays a role in the pathogenesis of ALS without VAPB mutations.

Results

To investigate the pathogenic mechanism in vivo, we generated human wild type (wtVAPB) and mutant VAPB (muVAPB) transgenic mice that expressed the transgenes broadly in the CNS. We observed robust VAPB-positive aggregates in the spinal cord of muVAPB transgenic mice. However, we failed to find an impairment of motor function and motor neuron degeneration. We also did not detect any change in the endogenous VAPB level or evidence for induction of the unfolded protein response (UPR) and coaggregation of VAPA with muVAPB. Furthermore, we crossed these VAPB transgenic mice with mice that express mutant SOD1G93A and develop motor neuron degeneration. Overexpression of neither wtVAPB nor muVAPB modulated the protein aggregation and disease progression in the SOD1G93A mice.

Conclusion

Overexpression of VAPBP56S mutant to approximately two-fold of the endogenous VAPB in mouse spinal cord produced abundant VAPB aggregates but was not sufficient to cause motor dysfunction or motor neuron degeneration. Furthermore, overexpression of either muVAPB or wtVAPB does not modulate the course of ALS in SOD1G93A mice. These results suggest that changes in wild type VAPB do not play a significant role in ALS cases that are not caused by VAPB mutations. Furthermore, these results suggest that muVAPB aggregates are innocuous and do not cause motor neuron degeneration by a gain-of-toxicity, and therefore, a loss of function may be the underlying mechanism.     

Conformational stability of ribonuclease T1 determined by hydrogen-deuterium exchange.

The hydrogen-deuterium exchange kinetics of 37 backbone amide residues in RNase T1 have been monitored at 25, 40, 45, and 50 degrees C at pD 5.6 and at 40 and 45 degrees C at pD 6.6. The hydrogen exchange rate constants of the hydrogen-bonded residues varied over eight orders of magnitude at 25 degrees C with 13 residues showing exchange rates consistent with exchange occurring as a result of global unfolding. These residues are located in strands 2-4 of the central beta-pleated sheet. The residues located in the alpha-helix and the remaining strands of the beta-sheet exhibited exchange behaviors consistent with exchange occurring due to local structural fluctuations. For several residues at 25 degrees C, the global free energy change calculated from the hydrogen exchange data was over 2 kcal/mol greater than the free energy of unfolding determined from urea denaturation experiments. The number of residues showing this unexpected behavior was found to increase with temperature. This apparent inconsistency can be explained quantitatively if the cis-trans isomerization of the two cis prolines, Pro-39 and Pro-55, is taken into account. The cis-trans isomerization equilibrium calculated from kinetic data indicates the free energy of the unfolded state will be 2.6 kcal/mol higher at 25 degrees C when the two prolines are cis rather than trans (Mayr LM, Odefey CO, Schutkowski M, Schmid FX. 1996. Kinetic analysis of the unfolding and refolding of ribonuclease T1 by a stopped-flow double-mixing technique. Biochemistry 35: 5550-5561). The hydrogen exchange results are consistent with the most slowly exchanging hydrogens exchanging from a globally higher free energy unfolded state in which Pro-55 and Pro-39 are still predominantly in the cis conformation. When the conformational stabilities determined by hydrogen exchange are corrected for the proline isomerization equilibrium, the results are in excellent agreement with those from an analysis of urea denaturation curves.     

Restricted backbone conformational and motional flexibilities of loops containing peptidyl-proline bonds dominate the enzyme activity of staphylococcal nuclease

The role of cis-trans isomerizations of peptidyl-proline bonds in the enzyme activity of staphylococcal nuclease (SNase) was examined by mutation of proline residues. The proline-free SNase ([Pro-]SNase), namely, P11A/P31A/P42A/P47T/P56A/P117G-mutant SNase, was adopted for elucidating the correlation between the nuclease activity and the backbone conformational and dynamic states of SNase. The 3D solution structure of [Pro-]SNase has been determined by heteronuclear NMR experiments. Comparing the structure of [Pro-]SNase with the structure of SNase revealed the conformational differences between the two proteins. In the structure of [Pro-]SNase, conformational rearrangements were observed for the loop of residues Ala112-His121 containing a trans Lys116-Gly117 peptide bond and for the C-terminal alpha-helical loop of residues Leu137-Glu142. Mutation of proline at position 117 also caused the conformational rearrangement of the p-loop (Asp77-Leu89), which is remote from the Ala112-His121 loop. The Ala112-His121 loop and p-loop are placed closer to each other in [Pro-]SNase than in SNase. The backbone dynamic features of the omega-loop (Pro42-Pro56) of SNase are different from those of [Pro-]SNase. The backbone of the omega-loop exhibits restricted flexibility with slow conformational exchange motions in SNase, but is highly flexible in [Pro-]SNase. The analysis indicates that the restrained backbone conformation of the Ala112-His121 loop and restricted flexibility of the omega-loop are two dominant factors determining the enzyme activity of SNase. Of the two factors, the former is correlated with the strained cis Lys116-Pro117 peptide bond and the latter is correlated with the cis-trans isomerizations of the His46-Pro47 peptide bond.     

Structural and functional relations among thioredoxins of different species

Three-dimensional models have been constructed of homologous thioredoxins and protein disulfide isomerases based on the high resolution x-ray crystallographic structure of the oxidized form of Escherichia coli thioredoxin. The thioredoxins, from archebacteria to humans, have 27-69% sequence identity to E. coli thioredoxin. The models indicate that all the proteins have similar three-dimensional structures despite the large variation in amino acid sequences. As expected, residues in the active site region of thioredoxins are highly conserved. These include Asp-26, Ala-29, Trp-31, Cys-32, Gly-33, Pro-34, Cys-35, Asp-61, Pro-76, and Gly-92. Similar residues occur in most protein disulfide isomerase sequences. Most of these residues form the surface around the active site that appears to facilitate interactions with other enzymes. Other structurally important residues are also conserved. A proline at position 40 causes a kink in the alpha-2 helix and thus provides the proper position of the active site residues at the amino end of this helix. Pro-76 is important in maintaining the native structure of the molecule. In addition, residues forming the internal contact surfaces between the secondary structural elements are generally unchanged such as Phe-12, Val-25, and Phe-27.     

The transmembrane prolines of the mitochondrial ADP/ATP carrier are involved in nucleotide binding and transport and its biogenesis

The mitochondrial ADP/ATP carrier (Ancp) is a paradigm of the mitochondrial carrier family, which allows cross-talk between mitochondria, where cell energy is mainly produced, and cytosol, where cell energy is mainly consumed. The members of this family share numerous structural and functional characteristics. Resolution of the atomic structure of the bovine Ancp, in a complex with one of its specific inhibitors, revealed interesting features and suggested the involvement of some particular residues in the movements of the protein to perform translocation of nucleotides from one side of the membrane to the other. They correspond to three prolines located in the odd-numbered transmembrane helices (TMH), Pro-27, Pro-132, and Pro-229. The corresponding residues of the yeast Ancp (Pro-43, Ser-147, and Pro-247) were mutated into alanine or leucine, one at a time and analysis of the various mutants evidenced a crucial role of Pro-43 and Pro-247 during nucleotide transport. Beside, replacement of Ser-147 with proline does not inactivate Ancp and this is discussed in view of the conservation of the three prolines at equivalent positions in the Ancp sequences. These prolines belong to the signature sequences of the mitochondrial carriers and we propose they play a dual role in the mitochondrial ADP/ATP carrier function and biogenesis. Unexpectedly their mutations cause more general effects on mitochondrial biogenesis and morphology, as evidenced by measurements of respiratory rates, cytochrome contents, and also clearly highlighted by fluorescence microscopy.     

Characterization of the properties of a novel mutation in VAPB in familial amyotrophic lateral sclerosis

Following the mutation screening of genes known to cause amyotrophic lateral sclerosis (ALS) in index cases from 107 familial ALS (FALS) kindred, a point mutation was identified in vesicle-associated membrane protein-associated protein B (VAPB), or VAMP-associated protein B, causing an amino acid change from threonine to isoleucine at codon 46 (T46I) in one FALS case but not in 257 controls. This is an important finding because it is only the second mutation identified in this gene that causes ALS. In order to investigate the pathogenic effects of this mutation, we have used a motor neuron cell line and tissue-specific expression of the mutant protein in Drosophila. We provide substantial evidence for the pathogenic effects of this mutation in abolishing the effect of wild type VAPB in the unfolded protein response, promoting ubiquitin aggregate formation, and activating neuronal cell death. We also report that expression of the mutant protein in the Drosophila motor system induces aggregate deposition, endoplasmic reticulum disorganization, and chaperone up-regulation both in neurons and in muscles. Our integrated analysis of the pathogenic effect of the T46I mutation and the previously identified P56S mutation indicate extensive commonalities in the disease mechanism for these two mutations. In summary, we show that this newly identified mutation in human FALS has a pathogenic effect, supporting and reinforcing the role of VAPB as a causative gene of ALS.     

Inheritance of cortical ER in yeast is required for normal septin organization

How cells monitor the distribution of organelles is largely unknown. In budding yeast, the largest subdomain of the endoplasmic reticulum (ER) is a network of cortical ER (cER) that adheres to the plasma membrane. Delivery of cER from mother cells to buds, which is termed cER inheritance, occurs as an orderly process early in budding. We find that cER inheritance is defective in cells lacking Scs2, a yeast homologue of the integral ER membrane protein VAP (vesicle-associated membrane protein-associated protein) conserved in all eukaryotes. Scs2 and human VAP both target yeast bud tips, suggesting a conserved action of VAP in attaching ER to sites of polarized growth. In addition, the loss of either Scs2 or Ice2 (another protein involved in cER inheritance) perturbs septin assembly at the bud neck. This perturbation leads to a delay in the transition through G2, activating the Saccharomyces wee1 kinase (Swe1) and the morphogenesis checkpoint. Thus, we identify a mechanism involved in sensing the distribution of ER.     

The structure of a monomeric mutant Cks protein reveals multiple functions for a conserved hinge-region proline

Cks (cyclin-dependent kinase subunit) proteins are essential eukaryotic cell cycle regulatory proteins that physically associate with cyclin-dependent kinases (Cdks) to modulate their activity. Cks proteins have also been studied for their ability to form domain-swapped dimers by exchanging β-strands. Domain swapping is mediated by a conserved β-hinge region containing two proline residues. Previous structural studies indicate that Cks in its dimer form is unable to bind Cdk, suggesting that the monomer-dimer equilibrium of Cks may have an effect on Cks-mediated Cdk regulation. We present the crystal structure of a proline-to-alanine mutant Saccharomyces cerevisiae Cks protein (Cks1 P93A) that preferentially adopts the monomer conformation but surprisingly fails to bind Cdk. Comparison of the Cks1 P93A structure to that of other Cks proteins reveals that Pro93 is critical for stabilizing a multiple β-turn structure in the hinge region that properly positions an essential Cdk-binding residue. Additionally, we find that these β-turn formations, conserved in Cks homologs, have implications for the mechanism and preferentiality of strand exchange. Together, our observations suggest that the conservation of Cks hinge-region prolines reflects their functions in forming a Cdk binding interface and that the ability of these prolines to control partitioning between monomer and dimer is a consequence of the β-turn networks within the hinge.